Application Verification Testing for Western Blot

The typical western blot protocol that Invitrogen antibodies are subjected to is reproduced below. This protocol includes:

• Sample preparation
• SDS-PAGE
• Transfer
• Membrane staining
• Blocking
• Probing with primary and secondary antibodies
• Developing the blot

Note: Some of the steps in this protocol require optimization depending on the sample and antibody being used.

Sample preparation

1. Prepare the samples to be loaded at desired concentration in lysis buffer and LDS sample buffer (with or without reducing agent) by boiling for 10 min at 100^oC

Note: Typically, 10–50 µg of protein is loaded per well depending on protein abundance, molecular weight, and type of gel used.

2. Let samples cool and spin briefly to collect any precipitate. 

SDS-PAGE

1. Assemble SDS-PAGE gel system and fill with SDS PAGE running buffer.
   
2. Remove comb from gel. Make sure the wells are free from bubbles or residual polyacrylamide.
   
3. Load the samples and appropriate molecular weight marker.

Note: The molecular weight (MW) marker should cover the expected MW range to ensure the MW can be accurately estimated after the detection step.

4. Run gels in a SDS-PAGE gel system (i.e., XCell SureLock apparatus) at up to 150 volts. Make sure the running buffer is not leaking and that the system remains cool so the gel does not melt.
   
5. Observe the loading dye front and stained molecular weight marker to determine how long to run the gel. 

Transfer  

Wet transfer (suggested for high–molecular weight and very low-abundance proteins)

1. Prepare transfer apparatus by soaking all sponges and filter paper in transfer buffer.
   
2. Remove the gel from the SDS-PAGE gel system, rinse with deionized water and equilibrate in transfer buffer.
   
3. Prepare the transfer stack in a blot module (i.e., XCell II Blot Module). The stack includes (from cathode to anode): 3 absorbent sponges, filter paper, gel, membrane, filter paper, and 3 absorbent sponges.

Note: When placing membrane over gel, make sure to remove all air bubbles.

4. Place the blot module in the XCell SureLock Mini-Cell and place wedge.
   
5. Fill core completely with transfer buffer and the outer chamber completely with deionized water.
   
6. Run at 80 volts for 2–3 hours or at 25 volts overnight in cold room.

Note: The time to transfer the protein will vary according to the molecular weight of the protein.

Dry transfer (suggested for proteins <100 kDa) with iBlot transfer system

1. Open the lid of the iBlot 3 Western Blot Transfer System using the latch. Ensure the blotting surface is clean.
   
2. Unseal the iBlot 3 transfer stack, separate the top stack and set it to the side with the transfer gel layer facing up. Keep the bottom stack in the transparent plastic tray.
   
3. Remove and discard the white separator from the top stack and place the bottom stack with the tray directly on the blotting surface.
   
4. Align the tray on the blotting surface according to the type of iBlot 3 transfer stack being used.

Note: The electrical contact on the tray should be aligned with the corresponding electrical contacts on the blotting surface of the iBlot 3 Western Blot Transfer Device.

5. Open the gel cassette and immerse the pre-run gel briefly in deionized water and place the gel on the transfer membrane of the bottom stack. Use the blotting roller to remove any air bubbles.
   
6. Soak the iBlot 3 filter paper in deionized water, and place the presoaked filter paper on the gel. Make sure to remove any air bubbles.
   
7. Place the top stack on top of the filter and gel with the copper electrode facing up. Remove any air bubbles.
   
8. Close the iBlot 3 Western Blot Transfer Device lid and choose appropriate transfer program.
   
9. Once the transfer is complete, remove the transfer membrane and proceed with the blocking or staining procedure.
   

Semi-dry transfer

1. In a shallow tray, briefly soak 2 stacked pieces of 2.5 mm thick blotting filter paper in transfer buffer.
   
2. Remove any air bubbles trapped between the filter paper by rolling the stack with the blotting roller while it is still submerged in buffer.
   
3. Place the stack of pre-soaked filter paper on the anode plates of the semi-dry blotter. Remove any air bubbles.
   
4. Place the pre-soaked blotting membrane on top of the filter paper and remove air bubbles.
   
5. Carefully remove the gel from the gel cassette and soak in transfer buffer.
   
6. Place the gel on top of the blotting membrane.
   
7. Briefly soak the remaining filter paper and stack on the gel. Ensure that the filter paper sheets are aligned properly and flush with the gel/membrane sandwich. Remove any air bubbles.
   
8. Place the cathode plate lid on the stack and tighten knobs.
   
9. Run at 20 volts for 30–60 min.

Membrane staining (optional)

To control for even transfer, membranes can be stained with ponceau-S using the following procedure:

1. Wash the membrane with 1X PBST (PBS-Tween 20) briefly and stain by adding ponceau-S stain solution on the membrane. Keep the membrane on gel rocker for 2 min.
2. Wash off excess stain by washing membrane with 1X PBST 3–4 times.

Blocking

Choose the blocking buffer that will work for your specific workflow. As with antibody concentration or detection substrates, the choice of blocking buffer can be critical for obtaining good results, and blocking should be optimized. In addition to the suggestions below, various ready-to-use blockers are available.

1. Block membrane with 5% skim milk or 5% BSA in 1X PBST or TBST (TBS-Tween 20) for 1 hour on a gel rocker at room temperature.

Note: For phosphorylated targets we recommend blocking with 5% BSA.

Probing with primary and secondary antibody  

HRP Conjugate

1. Incubate membrane with appropriate dilutions of primary antibody prepared in blocking buffer overnight at 4^oC or for 2–3 hours at room temperature on a gel rocker.

Note: The amount of primary and secondary antibody will vary depending on the antibody being used and the samples you are running. A good starting point is to check the antibody product pages for recommended dilution ranges and from there titrate to find the optimal antibody dilution for the experiment.

2. After incubation, wash the membrane three times with 1X PBST or TBST for 5 min.
   
3. Incubate the membrane for 30–45 min with the recommended dilution of HRP-conjugated secondary antibody prepared in blocking buffer at room temperature on a gel rocker.
   
4. Remove secondary antibody and wash the membrane three times with 1X PBST or TBST for 5 min each on a gel rocker.
    

Fluorescent Conjugate

Note: It is recommended to filter-sterilize buffers for fluorescent western blot to avoid aggregates getting on the membrane, which may become fluorescent artifacts. 

1. Incubate membrane with appropriate dilutions of primary antibody prepared in blocking buffer overnight at 4°C or for 2–3 hours at room temperature on a gel rocker.
   

Note: If multiplexing, use primary antibodies from different species to prevent cross-reactivity.

Note: The amount of primary and secondary antibody will vary depending on the antibody being used and the samples you are running. A good starting point is to check the antibody product pages for recommended dilution ranges and from there titrate to find the optimal antibody dilution for the experiment.

2. After incubation, wash the membrane three times with 1X PBST or TBST for 5 min.
3. Incubate the membrane for 30–45 min with the recommended dilution of fluorescent-conjugated secondary antibody prepared in blocking buffer at room temperature on a gel rocker. Cover the blot container with aluminum foil to protect the fluorophore from light.

Note: If multiplexing, use a cross-adsorbed secondary antibody to reduce the chance of cross-reactivity. Also, choose distinct fluorophores to avoid cross-channel bleed through.

4. Remove secondary antibody and wash the membrane three times with 1X PBST or TBST for 5 min each on a gel rocker.

Developing the blot  

HRP western blot

1. Develop the blot using ECL Chemiluminescent Substrate for 2–5 min. Place the membrane in clear film and remove any excess substrate.

Note: If the exposure time is longer than a few min, consider using a more sensitive substrate, such as SuperSignal West Pico, SuperSignal West Dura or SuperSignal West Femto.

2. Acquire image using a chemiluminescence imager, such as the iBright Imaging System.  

Fluorescent western blot

1. Acquire image using a fluorescent imager.
   
2. Thoroughly clean imaging surface with 70% alcohol before use.
   
3. Place the blot on the imaging surface and ensure it is lying flat with no air bubbles.
   
4. If you are multiplexing, use the 800 nm channel for detection of less abundant proteins or weak targets. Use the 680 nm channel for detection of more abundant proteins or strong targets.