Western Blot Detection Support — Troubleshooting

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General

I ran my protein sample on one of your gels and the bands look non-distinct and smeary after western detection. What should I do?

Here are some suggestions:

Make sure that the correct amount of protein is loaded per lane - loading too much protein can cause smearing.
Bands will not be as well resolved in low percentage gels – try using a higher percentage gel.
This may be due to the antibody being too concentrated. We recommend following the manufacturer’s recommended dilution or determining the optimal antibody concentration

I am unable to visualize my protein bands after western detection. What is the problem?

Here are possible causes and solutions:

Cause	Solution
The primary antibody and secondary antibody are not compatible	Use a secondary antibody that was raised against the species in which the primary antibody was raised.
The primary antibody is too dilute	1) Use a more concentrated antibody solution. 2) Incubate longer (e.g., overnight) at 4 degrees C. 3) Use fresh antibody and keep in mind that each time an antibody solution is used, its effective antibody concentration decreases.
Something in your blocking buffer interferes with binding of the primary and/or secondary antibody	Try an alternate blocking buffer ± a mild surfactant like Tween™-20 (0.01–0.05% v/v). There are many blocking buffer recipes available, based on non-fat dry milk, BSA, normal serum, gelatin and mixtures of these and other materials. Note that BSA (1–5%) is considered the best blocker for nitrocellulose membranes. It is easy to check the efficacy of different blocking buffers by performing dot-blots.
The primary antibody does not recognize the protein in the species being tested	1) Evaluate primary antibodies by dot-blotting first to how well they react with your protein. 2) Check the immunogen sequence, if provided, and determine if it is found in your protein. 3) If no immunogen sequence is available, perform a PubMed/BLAST alignment to assess the degree of homology between your target protein and the protein against which the antibody was generated. Note that many antibodies against human proteins will also recognize the non-human primate version because there is usually a high degree of amino acid identity. In contrast, many antibodies against human proteins will not recognize the corresponding proteins from rodents (and vice versa). Remember that significant homology between sequences does not guarantee that the antibody will recognize your protein. 4) Always run the recommended positive control, if available.
Insufficient protein is bound to the membrane or the protein of interest is not abundant enough in the sample	1) Load at least 20–30 μg protein per lane on your gels (as a starting point), since proteins representing less than ~0.2% of the total protein are difficult to detect on western blots. 2) Use an enrichment step to increase the concentration of the target protein. For example, prepare two nuclear lysates prior to blotting nuclear proteins or perform an immunoprecipitation (IP) prior to SDS-PAGE. 3) Reduce the volume of cell extraction buffer used to lyse your cells or tissue. 4) Be sure to use freshly prepared protease inhibitors and phosphatase inhibitors, if needed, in your protein extraction buffer. 5) Run the recommended positive control, if available.
Poor or no transfer of the proteins to the membrane	1) Check the protein transfer efficiency with a reversible protein stain like Novex™ Reversible Membrane Protein Stain, ponceau S, amido black or use pre-stained molecular weight standards. 2) Verify that the transfer was performed with the correct electrical polarity. 3) Remember that proteins with basic pI values (e.g., histones) and high MW may not transfer well. 4) Remember that if your target protein has a low MW (≤10 kDa), it may transfer more quickly than expected. 5) If you are using PVDF membranes, make sure to pre-soak the membrane in methanol first before soaking it in transfer buffer. Note that methanol in transfer buffer increases protein binding to nitrocellulose, but omitting methanol can increase transfer efficiency of high MW proteins. 6) Low MW proteins may pass through the 0.45 μm pores in nitrocellulose membranes, so switch to NC with 0.2 or 0.1 μm pores instead.
Excessive washing or blocking of the membrane	1) Avoid over-washing the membrane. Extra washing will not allow you to visualize your protein of interest if there are other problems with your blot. 2) Avoid over-blocking by using high concentrations of the blocking buffer components or long incubation times. Too much blocking can prevent your antibodies from binding to your protein. Gelatin, in particular, can mask proteins on the blot, so avoid it, if possible. Milk can also mask proteins, so instead of using 5% milk in your blocking buffer, try using it at 0.5% instead, or remove it altogether. 3) Switch to a different blocking reagent and/or block the blot for less time.
Using the same solution of diluted primary antibody repeatedly	Use freshly-diluted antibody for each western blot because the effective concentration of a diluted antibody decreases each time it is re-used. Also, remember that dilute solutions of antibodies are less stable and may lose their activity rapidly.
The enzyme conjugated to your secondary antibody is not working	1) Make a fresh dilution of your secondary antibody conjugate each time you need it. Enzymes (and antibodies) may lose activity quickly in dilute solutions. 2) Omit sodium azide in buffers if you are using HRP-conjugated antibodies. 3) Avoid high heme concentrations (from blood contamination), which can interfere with HRP-based detection. 4) Avoid using phosphate in buffers with alkaline phosphatase-antibody conjugates because phosphate inhibits enzyme activity.
Your colorimetric or other detection reagent is old and inactive	1) Use fresh enzyme substrate for each experiment. 2) Don’t use ready-to-use substrate reagents if they have changed color on their own or if they have passed their expiration date. 3) Do not dilute substrate solutions unless instructed to do so in the product manual.

I used your MagicMark™ XP Western Protein Standard and obtained a very weak or almost no signal after the western detection. Can you please help me troubleshoot?

Here are some suggestions:

Verify that the detection reagents are working well. Optimize the antibody concentration to obtain best results.
Make sure that the amount of standard loaded on the gel is correct.
Optimize the transfer conditions (current, voltage, transfer time).
Enzyme-conjugated primary antibodies may not bind efficiently with the proteins in the MagicMark™ XP Western Protein Standard. We recommend using unconjugated primary antibody, followed by the addition of enzyme-conjugated secondary antibody.

Note: The Anti-myc-AP/HRP and Anti-V5-AP/HRP antibodies do not bind to MagicMark™ XP proteins.

I am getting very high background after western detection. Can you please offer some tips?

Here are possible causes and solutions:

Cause	Solution
Insufficient blocking or non-specific binding	We suggest trying our WesternBreeze™ Blocker/Diluent (Cat. No. WB7050).
Membrane is contaminated	Use only clean, new membranes. Wear clean gloves at all times and use forceps when handling membranes.
Higher intrinsic background with PVDF membranes	Switch to nitrocellulose membranes.
Nitrocellulose membrane not completely wetted	Follow instructions for pre-wetting the membrane.
Blot is overdeveloped	Follow recommended developing time and remove blot from substrate when signal - to -noise ratio is acceptable.
Insufficient washing	Follow recommended number of washes. In some cases, it may be necessary to increase the number or duration of washes.
Concentrated secondary antibody used	Determine optimal antibody concentration by performing a dot blot and dilute antibody as necessary.
Concentrated primary antibody used	Determine optimal antibody concentration by performing a dot blot and dilute antibody as necessary.

I am getting a lot of non-specific binding after western detection. Can you offer some tips?

Here are possible causes and solutions:

Cause	Solution
Membrane contaminated by fingerprints or keratin proteins	Wear clean gloves at all times and use forceps when handling membranes. Always handle membranes around the edges.
Concentrated secondary antibody used	Make sure the secondary antibody is diluted as recommended. If the background remains high, but with strong band intensity, decrease the concentration of the secondary antibody.
Concentrated Primary antibody used	Decrease the concentration of the primary antibody.
Affinity of the primary antibody for the protein standards	Check with the protein standard manufacturer for homologies with primary antibody.
Insufficient removal of SDS or weakly bound proteins from membrane after blotting	Follow instructions for membrane preparation before immunodetection.
Short blocking time or long washing time	Make sure that each step is performed for the specified amount of time.

After western detection, my membrane has a lot of spots. What could have gone wrong?

Here are possible causes and solutions:

Membrane blotting pads are dirty or contaminated. Soak pads with detergent and rinse thoroughly with purified water before use. Replace pads when they become worn or discolored.
Blocking was uneven. The incubation dish must be sufficiently big to allow thorough coverage of membrane. Shake or agitate during each step.

How can I reduce background on my PVDF western blots?

PVDF membranes require more stringent blocking steps. This can be achieved by increasing the concentration of the blocking agent 2–5 fold, increasing the blocking time, and performing the procedure at 37 degrees C. Blocking agents bind to unoccupied sites to prevent background staining and also to membrane-bound proteins, reducing non-specific interactions with the primary antibody. Examples of blocking agents are nonfat dry milk, BSA, and Casein.

iBind™ Western System

I used the iBind™ Western system and am getting high background. What should I do?

Here are possible causes and solutions:

Cause	Solution
Membrane not completely wet	Follow instructions for prewetting the membrane. Use an incubation dish which is small enough to allow thorough coverage of membrane to prevent drying out. Note: If PVDF membrane is being used, we recommend making sure that it is activated with methanol, especially if it has dried up. It is not necessary to activate a PVDF membrane that has just come out of the transfer and moved into 1X iBind™ Solution/ iBind™ FD Solution.
Membrane is contaminated	Use only clean, new membranes. Wear clean gloves at all times and use forceps when handling membranes.
Film overexposed or became wet during exposure	Decrease exposure time or allow signal to further decay. Prevent leakage by encasing membrane in transparency film and blotting excess substrate from edges before exposure.
Solutions or incubation tray are contaminated	Use clean glassware and purified water to prepare solutions. Replace or clean the tray thoroughly with a glassware-cleaning detergent. Rinse thoroughly with purified water. Wear clean gloves at all times.
Concentrated primary antibody used	Follow the instructions provided in the manual to dilute the primary antibody or determine the optimum concentration by dot blotting.
Incorrect chemiluminescent substrate used for PVDF	Make sure CDP-Star™ reagent without enhancer is used.
Blot is overdeveloped	Follow recommended developing time or remove blot from substrate when signal-to-noise ratio is acceptable.
Ink used to label membrane	Any labeling of the membrane with ink should be limited to the low MW region of the blot.
Improper preparation of iBind™ Solution/ iBind™ FD Solution	Prepare 1X iBind™ Solution/ iBind™ FD Solution as directed in the manual.
Improper application of solutions to iBind™ Wells	Add the appropriate solutions for each well in the correct numerical order as specified on Page 18 in the manual.
Blot improperly placed on iBind™ Card	-Place the membrane in the designated Membrane Region on the iBind™ Card. -The protein side of the blot should be in contact with the iBind™ Card. -The low MW regions should be closest to the Stack. -The membrane should not be in contact with the Stack.

Here are some additional tips to reduce background:

If iBlot™ PVDF stacks are used, check that they have not expired as background increases with age. Using the iBlot™ 2 PVDF stacks will help in reducing the background.
Make sure that the blot is rinsed in distilled water prior to adding the substrate. Do not rinse in PBS or TBS.

With the iBind™ Western System, I am getting a lot of non-specific binding. Can you offer some tips?

Here are possible causes and solutions:

Cause	Solution
Membrane contaminated by fingerprints or keratin proteins	Wear clean gloves at all times and use forceps when handling membranes. Always handle membranes around the edges.
Primary antibody too concentrated	Follow the supplier’s recommended dilution or determine the optimum concentration by dot blotting.
Insufficient removal of SDS/weakly bound proteins from membrane after blotting	Follow instructions for membrane preparation before immunodetection, as directed in the manual.
Affinity of the primary antibody for the protein standards	Check with protein standard manufacturer for homologies with primary antibody.
Improper preparation of iBind™ Solution/ iBind™ FD Solution	Prepare 1X iBind™ Solution/ iBind™ FD Solution as directed in the manual.

I used the iBind™ Western System and am getting a very weak/almost no signal. What is the problem?

Here are possible causes and solutions:

Cause	Solution
Poor or incomplete transfer	Repeat blot. After blotting, stain membrane to measure transfer efficiency. Use positive control and/or molecular weight marker.
Membrane not completely wet	Follow instructions for prewetting the membrane.
Primary antibody concentration too low	Follow the supplier's recommended dilution or determine the optimum concentration by dot blotting.
Inactive primary antibody	Determine activity by performing a dot-blot.
Low affinity of primary antibody to antigen	Obtain a higher affinity primary antibody.
Contaminated secondary antibody solution	Wear gloves at all times and keep bottles tightly capped when not in use. Use only purified water when preparing reagents.
Protein of interest ran off the gel	Match gel separation range to size of protein being transferred.
Poor retention of proteins	Match gel separation range to size of protein being transferred. Use a molecular weight marker with relevant size proteins. Larger proteins require more transfer time, smaller proteins less. Use membrane with the appropriate binding capacity.
Improper preparation of iBind™ Solution/ iBind™ FD Solution	Prepare 1X iBind™ Solution/ iBind™ FD Solution as directed in the manual.
Improper application of solutions to iBind™ Wells	Ensure that the solutions are added to the correct wells and that the wells are loaded in numerical order.
Blot improperly placed on iBind™ Card	Ensure that the protein side of the blot is in contact with the iBind™ Card and is placed in the region labeled “membrane”.
Stack wet prior to run	Ensure that 5 mL of 1X iBind™ Solution/ iBind™ FD Solution is added to the flat region of the iBind™ Card. Avoid adding solution to the Stack.
Cross-contamination of solutions in wells	Do not move the iBind™ Western Device during the run.
iBind™ Card damaged	Replace with new card. Ensure that rolling of the membrane on the card is limited to the area labeled “membrane”.
Membrane is not in proper contact with the iBind™ Card	Place the membrane on the iBind™ Card immediately after adding a 1 mL pool of 1X iBind™ Solution/ iBind™ FD Solution. Use the roller provided to ensure proper contact.
Device opened prior to completion of run	The device should not be opened once the card has been placed in the device. Re-sealing of the wells on the card can result in leaks.
Sample improperly prepared; antigenicity weakened or destroyed	SDS and reducing agents may interfere with some antibody/antigen affinities.
Sample too dilute	Load a higher concentration or amount of protein onto the gel.
Protein weakly bound to membrane	Ensure that transfer buffer contains 10–20% methanol.
Insufficient exposure time	Re-expose film for a longer period of time.
Insufficient substrate incubation	Perform each step for the specified amount of time or remove blot from substrate when signal-to-noise ratio is acceptable.
Substrate is contaminated	Wear gloves at all times and keep bottles tightly capped when not in use.
Blots are too old	Protein may have broken down over time. Use freshly prepared blots.

I used the iBind™ Western System and have got a large, scattered signal. What could have caused this?

Here are possible causes and solutions:

Cause	Solution
Protein is overloaded	Reduce load or dilute concentration of sample.
Poor or incomplete transfer	Repeat blot.
Primary antibody is too concentrated	Follow the supplier's recommended dilution or determine the optimum concentration by dot blotting.

With the iBind™ Western system, lately, my run time has been in excess of 3 hours. What is the problem?

Here are possible causes and solutions:

Cause	Solution
iBind™ Card damaged	Replace with new card. Ensure that rolling of the membrane on the card is limited to the area labeled “membrane”.
Stack wet prior to run	Ensure that 5 mL of 1X iBind™ Solution/ iBind™ FD Solution is added to the flat region of the iBind™ Card. Avoid adding solution to the Stack.
Improper preparation of iBind™ Solution/ iBind™ FD Solution	Prepare 1X iBind™ Solution/ iBind™ FD Solution as directed in the manual.

I used the iBind™ Western system and my membrane is very “spotted”. What could have caused this?

Here are possible causes and solutions:

Cause	Solution
Poor or incomplete transfer	Repeat blot.
Membrane pads are dirty or contaminated	Soak with detergent and rinse thoroughly with purified water before use. Replace pads when they become worn or discolored.
Membrane not completely wet	Follow instructions for prewetting the membrane.
Membrane is contaminated by fingerprints or keratin proteins	Wear clean gloves at all times and use forceps when handling membranes. Always handle membranes around the edges.
Uneven blocking	The incubation dish must be small enough to allow thorough coverage of membrane.
Ink used to label membrane	Any labeling of the membrane with ink should be limited to the low MW region of the blot.
iBind™ Card damaged	Replace with new card. Ensure that rolling of the membrane on the card is limited to membrane region.
Membrane is not in proper contact with the iBind™ Card	Place the membrane on the iBind™ Card immediately after adding a 1 mL pool of 1X iBind™ Solution/ iBind™ FD Solution. Use the roller provided to ensure proper contact.

iBind™ Flex Western System

I used the iBind™ Flex Western system and am getting high background. What should I do?

Here are possible causes and solutions:

Cause	Solution
Membrane not completely wet	Follow instructions for prewetting the membrane. Use an incubation dish which is small enough to allow thorough coverage of membrane to prevent drying out. Note: If PVDF membrane is being used, we recommend making sure that it is activated with methanol, especially if it has dried up. It is not necessary to activate a PVDF membrane that has just come out of the transfer and moved into 1X iBind™ Flex Solution/ iBind™ Flex FD Solution.
Membrane is contaminated	Use only clean, new membranes. Wear clean gloves at all times and use forceps when handling membranes.
Film overexposed or became wet during exposure	Decrease exposure time or allow signal to further decay. Prevent leakage by encasing membrane in transparency film and blotting excess substrate from edges before exposure.
Solutions or incubation tray are contaminated	Use clean glassware and purified water to prepare solutions. Replace or clean the tray thoroughly with a glassware-cleaning detergent. Rinse thoroughly with purified water. Wear clean gloves at all times.
Concentrated primary antibody used	Follow the supplier's recommended dilution or determine the optimum concentration by dot blotting.
Incorrect chemiluminescent substrate used for PVDF	Make sure CDP-Star™ reagent without enhancer is used.
Blot is overdeveloped	Follow recommended developing time or remove blot from substrate when signal-to-noise ratio is acceptable.
Ink used to label membrane	Any labeling of the membrane with ink should be limited to the low MW region of the blot.
Improper preparation of iBind™ Flex Solution/ iBind™ Flex FD Solution	Prepare 1X iBind™ Flex Solution/ iBind™ Flex FD Solution as directed in the manual.
Improper application of solutions to iBind™ Flex Wells	Add the appropriate solutions for each well in the correct numerical order as specified in the manual.
Blot improperly placed on iBind™ Flex Card	-Place the membrane in the designated Membrane Region on the iBind™ Flex Card. -The protein side of the blot should be in contact with the iBind™ Flex Card. -The low MW regions should be closest to the Stack. -The membrane should not be in contact with the Stack.
Card stack wet prior to run	Ensure that 10 mL of 1X iBind™ Flex/iBind™ Flex FD Solution is added to the flow region of the card. Avoid adding the solution to the stack.

Here are some additional tips to reduce background:

If iBlot™ PVDF stacks are used, check that they have not expired as background increases with age. Using the iBlot™ 2 PVDF stacks will help in reducing the background.
Make sure that the blot is rinsed in distilled water prior to adding the substrate. Do not rinse in PBS or TBS.

With the iBind™ Flex Western System, I am getting a lot of non-specific binding. Can you offer some tips?

Here are possible causes and solutions:

Cause	Solution
Membrane contaminated by fingerprints or keratin proteins	Wear clean gloves at all times and use forceps when handling membranes. Always handle membranes around the edges.
Primary antibody too concentrated	Follow the supplier’s recommended dilution or determine the optimum concentration by dot blotting.
Insufficient removal of SDS/weakly bound proteins from membrane after blotting	Follow instructions for membrane preparation before immunodetection, as directed in the manual.
Affinity of the primary antibody for the protein standards	Check with protein standard manufacturer for homologies with primary antibody.
Improper preparation of iBind™ Flex Solution/ iBind™ Flex FD Solution	Prepare 1X iBind™ Flex Solution/ iBind™ Flex FD Solution as directed in the manual.

I used the iBind™ Flex Western System and am getting a very weak/almost no signal. What is the problem?

Here are possible causes and solutions:

Cause	Solution
Poor or incomplete transfer	Repeat blot. After blotting, stain membrane to measure transfer efficiency. Use positive control and/or molecular weight marker.
Membrane not completely wet	Follow instructions for prewetting the membrane.
Primary antibody concentration too low	Follow the supplier's recommended dilution or determine the optimum concentration by dot blotting.
Inactive primary antibody	Determine activity by performing a dot-blot.
Low affinity of primary antibody to antigen	Obtain a higher affinity primary antibody.
Contaminated secondary antibody solution	Wear gloves at all times and keep bottles tightly capped when not in use. Use only purified water when preparing reagents.
Protein of interest ran off the gel	Match gel separation range to size of protein being transferred.
Poor retention of proteins	Match gel separation range to size of protein being transferred. Use a molecular weight marker with relevant size proteins. Larger proteins require more transfer time, smaller proteins less. Use membrane with the appropriate binding capacity.
Improper preparation of iBind™ Flex Solution/ iBind™ Flex FD Solution	Prepare 1X iBind™ Flex Solution/ iBind™ Flex FD Solution as directed in the manual.
Improper application of solutions to iBind™ Flex Wells	Ensure that the solutions are added to the correct wells and that the wells are loaded in numerical order.
Blot improperly placed on iBind™ Flex Card	Ensure that the protein side of the blot is in contact with the iBind™ Flex Card and is placed in the region labeled “membrane”.
Stack wet prior to run	Ensure that 5 mL of 1X iBind™ Flex Solution/ iBind™ Flex FD Solution is added to the flat region of the iBind™ Flex Card. Avoid adding solution to the Stack.
Cross-contamination of solutions in wells	Do not move the iBind™ Flex Western Device during the run.
iBind™ Flex Card damaged	Replace with new card. Ensure that rolling of the membrane on the card is limited to the area labeled “membrane”.
Membrane is not in proper contact with the iBind™ Flex Card	Place the membrane on the iBind™ Flex Card immediately after adding a 1 mL pool of 1X iBind™ Flex Solution/ iBind™ Flex FD Solution. Use the roller provided to ensure proper contact.
Device opened prior to completion of run	The device should not be opened once the card has been placed in the device. Re-sealing of the wells on the card can result in leaks.
Sample improperly prepared; antigenicity weakened or destroyed	SDS and reducing agents may interfere with some antibody/antigen affinities.
Sample too dilute	Load a higher concentration or amount of protein onto the gel.
Protein weakly bound to membrane	Ensure that transfer buffer contains 10–20% methanol.
Insufficient exposure time	Re-expose film for a longer period of time.
Insufficient substrate incubation	Perform each step for the specified amount of time or remove blot from substrate when signal-to-noise ratio is acceptable.
Substrate is contaminated	Wear gloves at all times and keep bottles tightly capped when not in use.
Blots are too old	Protein may have broken down over time. Use freshly prepared blots.

I used the iBind™ Flex Western System and have got a large, scattered signal. What could have caused this?

Here are possible causes and solutions:

Cause	Solution
Protein is overloaded	Reduce load or dilute concentration of sample.
Poor or incomplete transfer	Repeat blot.
Primary antibody is too concentrated	Follow the supplier's recommended dilution or determine the optimum concentration by dot blotting.

With the iBind™ Flex Western system, lately, my run time has been in excess of 3 hours. What is the problem?

Here are possible causes and solutions:

Cause	Solution
iBind™ Flex Card damaged	Replace with new card. Ensure that rolling of the membrane on the card is limited to the area labeled “membrane”.
Stack wet prior to run	Ensure that 5 mL of 1X iBind™ Flex Solution/ iBind™ Flex FD Solution is added to the flat region of the iBind™ Flex Card. Avoid adding solution to the Stack.
Improper preparation of iBind™ Flex Solution/ iBind™ Flex FD Solution	Prepare 1X iBind™ Flex Solution/ iBind™ Flex FD Solution as directed in the manual.

I used the iBind™ Flex Western system and my membrane is very “spotted”. What could have caused this?

Here are possible causes and solutions:

Cause	Solution
Poor or incomplete transfer	Repeat blot.
Membrane pads are dirty or contaminated	Soak with detergent and rinse thoroughly with purified water before use. Replace pads when they become worn or discolored.
Membrane not completely wet	Follow instructions for prewetting the membrane.
Membrane is contaminated by fingerprints or keratin proteins	Wear clean gloves at all times and use forceps when handling membranes. Always handle membranes around the edges.
Uneven blocking	The incubation dish must be small enough to allow thorough coverage of membrane.
Ink used to label membrane	Any labeling of the membrane with ink should be limited to the low MW region of the blot.
iBind™ Flex Card damaged	Replace with new card. Ensure that rolling of the membrane on the card is limited to membrane region.
Membrane is not in proper contact with the iBind™ Flex Card	Place the membrane on the iBind™ Flex Card immediately after adding a 1 mL pool of 1X iBind™ Flex Solution/ iBind™ Flex FD Solution. Use the roller provided to ensure proper contact.

Chemiluminescent and Chromogenic Detection

What is the 54 kDa band observed in human tissue lysates when I use WesternBreeze™ AP Substrates?

A number of human alkaline phosphatases that could be consuming the WesternBreeze™ substrate have a molecular weight close to 50 kDa. These would be detected as a band of approximately 54 kDa. These proteins are known to dimerize, and can show as a band of approximately 110 kDa in native gels.

Why do I see more bands with the SuperSignal Chemiluminescent Substrates than I've seen in the past with other substrates?

Because these substrates are more sensitive than other chemiluminescent substrates you might detect low-abundance proteins that were not visible before. When using a more sensitive substrate, more careful optimization of blocking buffer steps and antibody concentrations is required.

With the SuperSignal Chemiluminescent Substrates, why is the entire film black or showing a reversed image?

The antibody concentration (primary and secondary) is too high. Use the antibody dilutions described in the product instructions. Most background will disappear when the proper blocking reagent and a HRP conjugate concentration are used.

iBlot™ Western Detection Kits

Sometimes I see high background on the membrane after using an iBlot™ Western Detection Kit. How can I eliminate it?

Here are possible causes and solutions:

Cause	Solution
Membrane is contaminated	Use only clean, new membranes. Wear clean gloves at all times and use forceps when handling membranes.
Higher intrinsic background with PVDF membranes	Switch to nitrocellulose membranes.
Nitrocellulose membrane not completely wetted	Follow instructions for pre-wetting the membrane.
Incorrect ratio between Diluent Additive and Antibody Diluent Solution	Make sure the proper amount of Diluent Additive is used for preparation of Antibody Diluent Mix (see Page 8 of the manual).
Blot is overdeveloped	Follow recommended developing time and remove blot from substrate when signal-to-noise ratio is acceptable.
Incorrect program was used	Use only program P9 for the iBlot™ Western Detection protocol.
Enhancer added to substrate when using PVDF membrane	Make sure Enhancer is not added to Chemiluminescent Substrate for PVDF membranes.
Insufficient washing	Follow recommended number of washes. In some cases, it may be necessary to increase the number or duration of washes.
Concentrated secondary antibody used	Make sure the secondary antibody is diluted as described in the manual. If the background remains high, but with strong band intensity, decrease the concentration of the secondary antibody.
Concentrated primary antibody used	Decrease the concentration of the primary antibody.

How can I improve the signal intensity when using the iBlot™ Western Detection Kits?

Here are possible causes and solutions for weak/no signal:

Cause	Solution
Poor or incomplete transfer	Check transfer conditions, and repeat blot. Use positive control and/or molecular weight marker.
Enhancer not added to substrate when using nitrocellulose membrane	Add Enhancer to Chemiluminescent Substrate for nitrocellulose membranes.
Nitrocellulose membrane not completely wetted, or PVDF membrane not completely reactivated	Follow instructions for pre-wetting or reactivating the membrane as described on Page 12 of the manual.
Secondary antibody concentration too low	Use the recommended secondary antibody concentrations described on Page 10 of the manual.
Primary antibody concentration too low	Use twice the concentration of primary antibody required for a standard immunodetection. If the signal is still low and the background is not high, increase the concentration.
Inactive primary antibody	Determine activity by performing a dot-blot or other methods.
Low affinity of primary antibody to antigen	Obtain a higher affinity primary antibody.
Sample improperly prepared; antigenicity weakened, or destroyed	SDS and reducing agents may interfere with some antibody/antigen affinities.
Sample too dilute	Load a higher concentration or amount of protein onto the gel.
Blots are too old	Protein may have broken down over time. Use freshly prepared blots.
Incorrect ratio between Diluent Additive and Antibody Diluent Solution	Make sure the proper amount of Diluent Additive is used for preparation of Antibody Diluent Mix (see Page 8 of the manual).
Protein of interest ran off the gel	Match gel separation range to size of protein being transferred.
Poor retention of proteins	Match the gel separation range to the size of the protein being transferred. Larger proteins require more transfer time, while smaller proteins require less transfer time. Use a molecular weight marker with relevant size proteins. Use membrane with the appropriate binding capacity.

I am getting non-specific binding with an iBlot™ Western Detection Kit. Can you offer some tips?

Cause	Solution
Membrane contaminated by fingerprints or keratin proteins	Wear clean gloves at all times and use forceps when handling membranes. Always handle membranes around the edges.
Concentrated secondary antibody used	Make sure the secondary antibody is diluted as described on Page 11 of the manual. If the background remains high, but with strong band intensity, decrease the concentration of the secondary antibody.
Concentrated Primary antibody used	Decrease the concentration of the primary antibody.
Affinity of the primary antibody for the protein standards	Check with the protein standard manufacturer for homologies with primary antibody.

I am using the iBlot™ Dry Blotting System with the iBlot™ Western Detection Kit and am getting the “Error2” message displayed. What is the issue?

Here are possible causes and solutions:

Short circuit or current exceeding the limits of the device. Open the lid for 15 seconds to allow the system to cool down. Close the lid and resume the run by pressing the Start/Stop Button. If the problem persists, contact Technical Support by e-mailing techsupport@lifetech.com.
Antibody Diluent Additive in the Antibody Diluent Mix exceeds 30% causing increased current and heat. Make sure the Antibody Diluent Mix is prepared as described on Page 8 of the manual.

Sometimes my molecular weight (MW) markers are no longer visible after my detection is completed with an iBlot™ Western Detection Kit. Does this mean my proteins are also gone?

No. Molecular weight markers are usually pre-stained and more highly charged than typical cellular proteins. Applying an electrical field in the western detection procedure can drive the MW markers right through the membrane onto the bottom stack.

Cellular proteins, however, are less charged than the molecular weight markers, and at the detection stage they no longer contain any SDS. The result is that the cellular proteins stay immobilized on the membrane during western detection using the iBlot™ system.

WesternDot™ Fluorescent Immunodetection

I am using a WesternDot™ secondary antibody conjugate for fluorescent Immunodetection and am getting a weak signal. What do I do to improve detection sensitivity?

Many factors may reduce signal intensity. One major factor is the primary antibody. Primary antibodies from different sources can have very different affinities to the target antigen. If weak signal is observed, try a primary antibody from a different vendor and /or from different host species, as this can help to improve signal intensity.

I was able to see a signal on my blots with a WesternDot™ conjugate originally, but the signal is gone after a few days. What happened?

WesternDot™ signals can fade when bound to dried PVDF membranes. For archiving stained blots or imaging after more than 24 hours, we recommend using nitrocellulose membranes.

I am observing a number of minor bands in my lysates after detection with the WesternDot™ Qdot™ 625 streptavidin conjugate supplied in the WesternDot™ 625 kits. Why is this?

There are a number of endogenous biotinylated proteins in cell lysate samples that will also be detected with streptavidin WesternDot™ conjugates. Endogenous biotinylated proteins can be confirmed by incubating a control blot in only the streptavidin WesternDot™ conjugate without the primary and secondary antibody. These endogenous biotinylated protein bands will be consistent in every lane containing the same cell lysate type and can serve as internal loading controls or can be blocked prior to western detection using the reagents in the Endogenous Biotin Blocking Kit (Cat. No. E21390).

My WesternDot™ reagent is showing some precipitate in the tube. Can I still use it?

If there is a large amount of precipitate, it likely indicates that the reagent has been frozen and should be discarded. Qdot™ probes will irreversibly aggregate at freezing temperatures. The formation of a small amount of precipitate during storage at 2–8 degrees C is normal. We recommend spinning the WesternDot™ reagent tube down briefly in a microcentrifuge with every use to remove any minor precipitate that has formed during storage and use only the supernatant.

I am getting high background after WesternDot™ detection. Can you please offer some tips?

Here are possible causes and solutions:

Cause	Solution
Insufficient blocking or non-specific binding	Try a different blocking reagent or increase the concentration of the blocking reagent. We generally obtain good results with 2% casein, 5% non-fat dry milk, or 1/2x fish serum.
Membrane was blocked with BSA	Do not use BSA-containing solutions for blocking or incubating WesternDot™ conjugates. For primary antibodies that are incompatible with casein or milk (e.g., many anti-phosphoprotein antibodies), use fish serum or use a 0.5% BSA-containing solution for primary antibody incubation only and then switch to 2% casein or 5% non-fat milk for all other incubation steps.
Membrane is contaminated	Use only clean, new membranes. Wear clean gloves at all times and use forceps when handling membranes.
Higher intrinsic background with PVDF membranes	Switch to nitrocellulose membranes.
Nitrocellulose membrane not completely wetted	Follow instructions for pre-wetting the membrane.
Insufficient washing	Follow recommended number of washes. In some cases, it may be necessary to increase the number or duration of washes.
Concentration of primary and/or secondary antibody is too high	Determine optimal antibody concentration by performing a dot blot and dilute antibody as necessary.

How can I improve the signal intensity when using WesternDot™ detection reagents?

Here are possible causes and solutions for weak/no signal:

Cause	Solution
Poor or incomplete transfer	Check transfer conditions, and repeat blot. Use positive control and/or molecular weight marker.
Nitrocellulose membrane not completely wetted, or PVDF membrane not completely reactivated	Follow instructions for pre-wetting or reactivating the membrane.
Secondary antibody concentration too low	Use the recommended secondary antibody concentrations.
Primary antibody concentration too low	Use twice the concentration of primary antibody required for a standard immunodetection. If the signal is still low and the background is not high, increase the concentration.
Inactive primary antibody	Determine activity by performing a dot-blot or other methods.
Low affinity of primary antibody to antigen	Obtain a higher affinity primary antibody.
Sample improperly prepared; antigenicity weakened, or destroyed	SDS and reducing agents may interfere with some antibody/antigen affinities.
Sample too dilute	Load a higher concentration or amount of protein onto the gel.
Blots are too old	Protein may have broken down over time. Use freshly prepared blots.
Protein of interest ran off the gel	Match gel separation range to the size of the protein being transferred.
Poor retention of proteins	Larger proteins require more transfer time, while smaller proteins require less transfer time. Use a molecular weight marker with relevant size proteins. Use membrane with the appropriate binding capacity.
WesternDot™ reagents have been frozen	Qdot™ probes will irreversibly aggregate at freezing temperatures

I am getting non-specific binding with my WesternDot™ detection reagent. Can you offer some tips?

Here are possible causes and solutions:

Cause	Solution
Membrane contaminated by fingerprints or keratin proteins	Wear clean gloves at all times and use forceps when handling membranes. Always handle membranes around the edges.
Concentrated secondary antibody used	Make sure the secondary antibody is diluted as recommended. If the background remains high, but with strong band intensity, decrease the concentration of the secondary antibody.
Concentrated Primary antibody used	Decrease the concentration of the primary antibody.
Affinity of the primary antibody for the protein standards	Check with the protein standard manufacturer for homologies with primary antibody.

Multicolored Fluorescent Western Detection—Alexa Fluor™ 680 and Alexa Fluor™ 790 Conjugates

I am getting high background with Alexa Fluor™ 790 and 680 secondary conjugates. What can I do to reduce background?

Add 0.1% SDS to blocker for secondary antibody incubation step to further reduce nonspecific background staining. Use lower fluorescent PVDF-FL membranes rather than PVDF.

I am getting high background after Alexa Fluor™ 790 and 680 detection. Can you please offer some tips?

Here are possible causes and solutions:

Cause	Solution
Insufficient blocking or non-specific binding	Try a different blocking reagent or increase the concentration of the blocking reagent. We generally obtain good results with 2% casein, 5% non-fat dry milk, or 1/2X fish serum.
Membrane was blocked with BSA	Do not use BSA-containing solutions for blocking or incubating Alexa Fluor™ 680 and 790 conjugates. For primary antibodies that are incompatible with casein or milk (e.g., many anti-phosphoprotein antibodies), use fish serum or use a 0.5% BSA-containing solution for primary antibody incubation only and then switch to 2% casein or 5% non-fat milk for all other incubation steps.
Membrane is contaminated	Use only clean, new membranes. Wear clean gloves at all times and use forceps when handling membranes.
Higher intrinsic background with PVDF membranes	Switch to nitrocellulose membranes.
Nitrocellulose membrane not completely wetted	Follow instructions for pre-wetting the membrane.
Insufficient washing	Follow recommended number of washes. In some cases, it may be necessary to increase the number or duration of washes.
Concentration of primary and/or secondary antibody is too high	Determine optimal antibody concentration by performing a dot blot and dilute antibody as necessary.

How can I improve the signal intensity when using Alexa Fluor™ 790 and 680 detection reagents?

Here are possible causes and solutions for weak/no signal:

Cause	Solution
Poor or incomplete transfer	Check transfer conditions, and repeat blot. Use positive control and/or molecular weight marker.
Nitrocellulose membrane not completely wetted, or PVDF membrane not completely reactivated	Follow instructions for pre-wetting or reactivating the membrane.
Secondary antibody concentration too low	Use the recommended secondary antibody concentrations.
Primary antibody concentration too low	Use twice the concentration of primary antibody required for a standard immunodetection. If the signal is still low and the background is not high, increase the concentration.
Inactive primary antibody	Determine activity by performing a dot-blot or other methods.
Low affinity of primary antibody to antigen	Obtain a higher affinity primary antibody.
Sample improperly prepared; antigenicity weakened, or destroyed	SDS and reducing agents may interfere with some antibody/antigen affinities.
Sample too dilute	Load a higher concentration or amount of protein onto the gel.
Blots are too old	Protein may have broken down over time. Use freshly prepared blots.
Protein of interest ran off the gel	Match gel separation range to the size of the protein being transferred.
Poor retention of proteins	Larger proteins require more transfer time, while smaller proteins require less transfer time. Use a molecular weight marker with relevant size proteins. Use membrane with the appropriate binding capacity.
Alexa Fluor™ 790 and 680 reagents have been repeatedly frozen	Repeated freeze/thawing can cause antibodies to irreversibly precipitate. For long-term storage, it is best to aliquot into individual use tubes before freezing.

I am getting non-specific binding with my Alexa Fluor™ 790 and 680 reagent. Can you offer some tips?

Here are possible causes and solutions:

Cause	Solution
Membrane contaminated by fingerprints or keratin proteins	Wear clean gloves at all times and use forceps when handling membranes. Always handle membranes around the edges.
Concentrated secondary antibody used	Make sure the secondary antibody is diluted as recommended. If the background remains high, but with strong band intensity, decrease the concentration of the secondary antibody.
Concentrated Primary antibody used	Decrease the concentration of the primary antibody.
Affinity of the primary antibody for the protein standards	Check with the protein standard manufacturer for homologies with primary antibody.

BenchPro™ 4100 Card Processing Station

I am using the BenchPro™ 4100 Card Processing Station and I don’t see any power meaning that the digital display remains blank and the pump does not run when the power is turned on. What should I do?

Here are possible causes and solutions:

AC power cord is not connected. Check AC power cord connections at both ends. Use the correct cords.
Fuse has blown. Replace the fuse (see page 31 in the manual). If the problem still persists after verifying that correct power cord is used and the fuse is replaced, contact Technical Support at techsupport@thermofisher.com.

As soon as I started using the BenchPro™ 4100 Card Processing Station, I got an error message. What went wrong?

Please refer to the list of error messages and actions to take on page 30 in the manual.

I am using the BenchPro™ 4100 Card Processing Station. What would happen if I accidentally missed adding card, reagents, or reagent bottles?

We recommend making sure that you have added the card, reagents, and reagent bottles in the correct order prior to starting the protocol. If the run is aborted after the run has begun, you need to restart the run. Do not reuse the card, if the run is aborted within a few minutes of starting the run.

I am finding it very difficult to insert the Western Card in the BenchPro™ 4100 Card Processing Station. What is the problem?

Here are possible causes and solutions:

Bottles have caps. Make sure that the reagent bottles and vials are uncapped when placed in the reagent tray.
The card is not oriented correctly. Insert the card into a slot in the BenchPro™ 4100 Card Processing Station such that the tips are inserted into the reagent bottles and vials placed in the reagent tray. Use the Card Alignment Guide to ensure the card is placed correctly in the slot. When the card is inserted correctly in the slot, you will hear a hard click.

I used the BenchPro™ 4100 Card Processing Station and got a very weak to almost no signal on my blot. Can you please help me troubleshoot?

Here are possible causes and solutions:

Cause	Solution
Detection step missed or detection reagents not working	The detection step is not included in the instrument protocol. After the instrument protocol is complete, perform the detection step using your standard detection reagents and protocol manually. Make sure the detection reagents are functional.
Insufficient incubation with detection reagent	Remove the blot from the detection reagent when signal-to-noise ratio is acceptable.
Incorrect order of reagents added	Be sure to add the reagents in the order described on page 13 of the manual to ensure all steps are performed correctly.
Poor or incomplete transfer	Make sure transfer apparatus and membrane sandwiches are assembled correctly. Use appropriate transfer times. After blotting, stain membrane to measure transfer efficiency.
Protein of interest ran off the gel	Use positive control and/or molecular weight marker to match gel separation range to size of protein being blotted. After blotting, stain membrane to measure transfer efficiency.
Sample was too dilute	Load a larger amount of protein onto the gel.
Poor retention of proteins or protein weakly bound to membrane	Ensure that transfer buffer contains 10–20% methanol. Use membranes with appropriate binding capacity.
Inactive or overly dilute primary or secondary antibody	Determine antibody activity by performing a dot blot. Increase antibody concentration as necessary.

I used the BenchPro™ 4100 Card Processing Station and got very high background on my blot. Can you offer some tips?

Here are possible causes and solutions:

Cause	Solution
Film overexposed or became wet during exposure	Decrease exposure time or allow signal to further decay. Prevent leakage of solutions by encasing membrane in transparency film and blotting excess substrate from edges before exposure.
Short blocking time or washing time	Perform each step for the specified amount of time.
High concentration of primary and/or secondary antibody	Determine optimal antibody concentration by performing a dot blot. Decrease antibody concentration as necessary.
Membrane, solutions, trays, or vials are contaminated	Use clean glassware and purified water to prepare solutions. Rinse the tray thoroughly with purified water. Although all reagent bottles and vials can be reused after washing with mild detergent and rinsing with deionized water, we do not recommend reusing the 25 ml vials to prevent any reagent cross-contamination. Wear clean gloves at all times. Use forceps when handling membranes.
Protein is overloaded	Reduce load or dilute the sample.

I used the BenchPro™ 4100 Card Processing Station and got non-specific binding. What happened?

Here are possible causes and solutions:

Cause	Solution
Insufficient removal of SDS or weakly bound proteins from membrane after blotting	Follow instructions for membrane preparation before immunodetection.
Short blocking time or long washing time	Make sure that each step is performed for the specified amount of time.
Affinity of the primary antibody for the protein standards	Check with protein standard manufacturer for homologies with primary antibody.
Membrane is contaminated by fingerprints or keratin proteins	Wear clean gloves at all times and use forceps when handling membranes. Always handle membranes around the edges.

In the BenchPro™ 4100 Card Processing Station, what could be the cause for no solution, or less than normal volume of solution, being pumped into the card blot chamber when a program is started?

Here are possible causes and solutions:

Cause	Solution
The Western Card may not be in the right position	Pull it out and insert it again to make sure it is inserted all the way down until you hear a click sound.
Solution intake tips could be clogged	Check your solutions, especially any milk blocking buffers, to see if there are large particles present that could be blocking the tips.
Defective cards	Try a new card, or a card from a box with a different lot number if possible. If you suspect a problem with your cards, please contact Technical Support at techsupport@lifetech.com .
Check instrument pressure and vacuum values	If numbers are significantly below 248 kPa for instrument pressure and below 48 kPa for vacuum, there could be a possible hardware issue. Please contact Technical Support at techsupport@lifetech.com .

With the BenchPro™ 4100 Card Processing Station, the LCD remains blank and the pump does not run when I turn on the power. What is the reason?

This could be due to the following reasons:

AC power cord is not connected or incorrect cord was used.
Fuse has blown.

When I started the BenchPro™ 4100 Card Processing Station, it 'warmed up' but the menu screen is not displayed as a result of which I am not able to enter my protocol. What should I do?

We would recommend shutting off the power and restarting the instrument.

With the BenchPro™ 4100 Card Processing Station, I am getting a high background upon detection using the LI-COR™ Imaging system. What do you recommend?

Here are some recommendations that may help:

Use a PBS-based Blocking reagent and do not use Tween™ 20 or BSA.
Make sure that the Primary and Secondary antibodies are freshly diluted in 0.01% Tween™ 20 in PBS
For the last 2 washes, use only PBS to get rid of the Tween™ 20, thus avoiding background.

The standard programs on the BenchPro™ 4100 end with a water rinse step. The membrane is held in this solution indefinitely so that it does not dry out. I want to create a custom program that ends with a wash step as I don’t want the final rinse step. If I do so, will my blot be left in the wash solution?

Yes, the blot will be held in the last reagent programmed, regardless which reagent that is. For the blot to be left in wash buffer, just make wash the last step instead of rinse.

iBright Imaging Systems

What should I do in case of a CCD cooling time issue with the iBright imager?

CCD cooling time can be impacted by room temperature and airing/ventilation mechanical blocking. Therefore, please first check the temperature in the room and potential blockage of the camera vent slots.

PowerBlotter

The power station on my G2 Fast Blotter has broken. How can I replace the station?

You can purchase the Power Blotter Station (Cat. No. PB0010) and the G2 Fast Blotter cassette is compatible with it. The new Power Blotter System is designed for Western transfer alone and does not have a staining module; therefore, the G2 Fast Blotter staining cassette cannot be used on the Power Blotter Station.

I have a G2 Fast Blotter but my cassette broke. Do you sell a spare cassette?

Yes, you can order the Power Blotter Cassette XL (Cat. No. PB0003).
Note: The Power Blotter Cassette (Cat. No. PB0002) is not compatible with the G2 Fast Blotter.

The Power Blotter Cassette/Power Blotter Cassette XL is difficult to open. What can I do?

This may be due to salt deposited on moving parts inside the cassette. We recommend rinsing the cassette top and bottom under warm water while removing any sticky salt residue with a gloved hand. Briefly rinse with deionized water and place in a rack to dry. For more thorough cleaning, immerse the unassembled cassette in warm water and use a gloved hand or clean sponge to remove any sticky salt residue. Rinse with deionized water and place perpendicular in a rack to dry.
Note: Failure to keep the cassette top (cathode) and bottom (anode) clean will result in the sticking of moving parts and lead to poor transfer efficiency.

I have lost the power cord for the Power Blotter Station. Is it sold as a stand-alone item?

Here are the SKUs for the power cord. Please order the one appropriate for your region:

- Cat. No. 84858: Power Cord with C13 Connector, North America
- Cat. No. 84859: Power Cord with C13 Connector, Continental Europe and South Korea
- Cat. No. 84860: Power Cord with C13 Connector, United Kingdom and Singapore
- Cat. No. 84855: Chinese 10 Amp Cordset w/ C13 Connector
- Cat. No. 84856: Japanese 15 Amp Cordset w/ C13 Connector
- Cat. No. 84857: Australian 10 Amp Cordset w/ C13 Connector

The central European or UK power cord for the Power Blotter System/Power Blotter XL System does not work in Switzerland. Do you offer a Swiss power cord?

Yes, you may purchase the Universal Cable Kit (Cat. No. 4377117).

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For Research Use Only. Not for use in diagnostic procedures.