Total protein normalization
Total protein normalization is a useful method for obtaining accurate, quantitative western blotting data. The Invitrogen No-Stain Protein Labeling Reagent is a fast, easy-to-use, covalent protein labeling reagent, applied to a gel or a membrane after protein transfer that provides sensitive, linear detection of protein for total protein normalization of western blotting data. The Invitrogen No-Stain Reagent can also be used as a fast, sensitive protein labeling reagent for visualization of proteins in a gel.
No-Stain Protein Labeling Reagent
- Versatile—Use for fast total protein labeling of gels and membranes. Compatible with all gel chemistries and with downstream gel staining, protein transfer, immunoblotting and mass spectrometry analysis.
- NEW Faster protocol—Mix, incubate and image. The reaction time is 10 minutes for gels and transferred membranes alike.
- Flexible visualization—Use a wide-range of excitation sources, including UV and green fluorescence light. Optimal imaging conditions: Ex. max: ~488 nm., Em. max: 590 nm.
- Accurate total protein normalization—Imaging and total protein normalization analysis take only minutes with the iBright FL1500 Imaging System
- Broad linear dynamic range—1–80 µg total protein loaded per well.
- Sensitive and stable signal—Protein bands are detected down to 20 ng and the signal is compatible with downstream antibody detection.
- Tunable sensitivity—Increasing incubation time or doubling the reagent concentration produces a proportionally stronger signal for improved detection of low abundance proteins.
How-to videos
How to use Invitrogen No-Stain Protein Labeling Reagent and the iBright Imaging System for fast, easy, and accurate quantitative western blotting
This video provides step-by-step instructions on how to label blotted proteins using No-Stain Protein Labeling Reagent. The second half of the video covers the on-instrument steps for total protein normalization of western blot data using the iBright Imaging System.
| Number of reactions | A standard kit will label 40 mini gel-sized membranes or 40 mini gels, or a combination of the two |
| Reaction time | 10 minutes |
| Gel compatibility | Compatible with any gel type (precast or pour your own) or gel chemistry (Bis-Tris, Tris-glycine, Tris-acetate, Tricine) |
| Membrane compatibility | PVDF, nitrocellulose |
| Immunodetection reagent compatibility | Compatible with all downstream immunodetection steps with chemiluminescence or fluorescence-based detection |
| Excitation and emission maxima | Excitation: ~488 nm (epi blue light) Emission: 590 nm |
| Detection sensitivity | 20 ng per band |
| Linear range | 1–80 μg total protein load per lane |
| Imager requirements | Compatible with a wide range of imagers that have UV or fluorescence light sources, for example, the iBright FL1500 Imaging System |
| How does it work? | The No-Stain Protein Labeling Reagent forms covalent bonds with a portion of the lysine amino acid side chains on all protein samples |
| Kit Component and storage conditions | No-Stain Activator (800 µL; storage -20⁰C); No-Stain Derivatizer (800 µL; storage -20⁰C); No-Stain Labeling Buffer, 20X (2 x 20 mL each; storage 15—30⁰C) |
| Shelf-life | At least 2 years |
No-Stain protocol for labeling protein on gels or transferred blots
Figure 1: No-Stain Protein Labeling Reagent Protocol
Preparation of No-Stain Protein Labeling Reagent
Mix the reagents needed to prepare the appropriate volume of No-Stain Protein Labeling Reagent as indicated in the table.
| No-Stain Protein Labeling Reagent Volume, 1X | No-Stain Labeling buffer, 20X | Ultrapure water | No-Stain Activator | No-Stain Derivatizer | |
|---|---|---|---|---|---|
| Mini gel | 20 mL | 1 mL | 19 mL | 20 µL | 20 µL |
| Mini membrane | 10 mL | 0.5 mL | 9.5 mL | 20 µL | 20 µL |
| Midi membrane | 20 mL | 1 mL | 19 mL | 40 µL | 40 µL |
In western blotting, variable results can be caused by unequal protein sample concentration, inconsistent sample loading onto the gel, and/or transfer variation during electroblotting. Protein normalization is used to correct for this variability and is a critical step in obtaining reliable and reproducible quantitative western blot data. Methods for protein normalization include use of internal housekeeping protein controls such as GAPDH, β-tubulin, β-actin, or cyclophilin B, exogenous loading controls, or total protein normalization.
In recent years, journal editors and reviewers have been requesting adoption of methods that improve the accuracy and reproducibility of quantitative western blot data. Many leading journals have developed guidelines for submitting western blotting research and select quotes from those guidelines are highlighted below.
- “For quantitative comparisons, appropriate reagents, controls and imaging methods with linear signal ranges should be used” – Nature
- “Record how data were obtained, whether signal intensity was linear with antigen loading, and how protein loading was normalized” – Journal of Biological Chemistry
- “Normalize signal intensity to total protein loading (assessed by staining membranes for total protein) whenever possible” – Journal of Biological Chemistry
- “House-keeping proteins should not be used for normalization without evidence that manipulations do not affect expression” – Journal of Biological Chemistry
The use of housekeeping proteins has its drawbacks as the expression of housekeeping gene proteins can vary with experimental conditions, and they can often have oversaturated western blotting signals.
Total protein normalization using the No-Stain Protein Labeling Reagent avoids the variability and inaccuracy of using housekeeping proteins and avoids the time, effort, and cost to detect the housekeeping gene protein using immunoblotting, which may involve stripping of the blot and re-probing.
An accurate loading control should display a linear relationship between signal intensity and sample load under all experimental conditions. The signal intensity obtained from labeling of total proteins on a membrane with No-Stain reagent ensures a linear relationship between signal intensity and sample load (Figure 2) in all experimental conditions. Therefore, the No-Stain reagent enables the use of total protein as an ideal loading control for quantitative western blotting applications.
Comparison of linearity of the No-Stain reagent to housekeeping proteins
The graph in Figure 2 below shows the linear signal response versus the amount of protein loaded per well using the No-Stain Protein Labeling Reagent for total protein normalization. Signals from the housekeeping proteins appear to be saturating at higher lysate loads and would provide less accurate normalization of results.
Figure 2. Total protein normalization using the No-Stain Protein Labeling Reagent: Bolt 4-12% Bis-Tris Plus gels were loaded with HeLa Lysate ranging from 10 to 50 µg and electrophoresed using MES running buffer. Proteins from the gels were transferred onto PVDF membranes using the Invitrogen iBlot 2 Gel Transfer Device with iBlot 2 Transfer Stacks, PVDF, mini (P0 protocol for 7 minutes). The PVDF membranes were washed twice for 2 minutes with 20 mL of ultra-pure water on a rotating platform, whereupon they were labeled with 10 mL of No-Stain labeling solution on a rotating platform for 10 minutes. The membranes were then washed 3 times for 2 minutes with 20 mL of ultra-pure water on a rotating platform, followed by immunoblotting for β-actin (Cat. no. AM4302), GAPDH (Cat. no. 398600), and α-tubulin (Cat. no. 138000) followed by goat anti-mouse Alexa Fluor Plus 680 (Cat. no. A21058). The blot was imaged using the iBright Imager. The iBright software was used to quantitate the total protein signal in the lanes. The linear regression value of the plotted data for the entire load range using the No-Stain Protein Labeling Reagent was determined (R2 = 0.9990), whereas the R2 values for β-actin, GAPDH, and α-tubulin were 0.8851, 0.9438, and 0.8332, respectively.
Figure 3 below illustrates normalization of a target protein using the No-Stain Protein Labeling Reagent with a nitrocellulose membrane.
Figure 3. Quantitative western blot analysis using the No-Stain Protein Labeling Reagent and an iBright Imaging System. A Novex 4-12% Tris-Glycine gel, WedgeWell format, was loaded and electrophoresed with lysates from HeLa cells expressing RB1, at total protein loads ranging from 0.6 to 10 µg. Proteins from the gel were transferred to a nitrocellulose membrane using the iBlot 2 Dry Blotting System. The nitrocellulose membrane was labeled using the No-Stain Protein Labeling Reagent for 10 minutes and the labeled membrane was imaged using the iBright imager. The same No-Stain labeled membrane was used to probe for RB1 with a specific antibody labeled with Alexa Fluor 645 dye. The iBright normalization software was used to quantify the total protein signal in lanes loaded with HeLa lysate loads ranging from 0.6 to 10 µg and signal intensities from RB1 immunodetection bands. The signal intensity from the total protein load and RB1 were plotted.
Labeling proteins in gels using the No-Stain Protein Labeling Reagent
The No-Stain Protein Labeling Reagent forms covalent bonds with a portion of the lysine amino acid side chains present in all proteins in a gel within 10 minutes.
- A great alternative to traditional gel staining reagents—No destaining is required and allows for accurate normalization over a wide range of protein lysate loads (1—80 µg)
- Sensitive—Lower limit of detection of 20 ng per band
- Specific—Forms bonds only with the lysine side chains of proteins. Free label does not fluoresce, thereby enabling a superior signal-to-noise ratio
- Flexible—Compatible with any gel type; no need to change your gels to get stain-free convenience
Figure 4 below shows labeling of protein on a blot with No-Stain Protein Labeling Reagent after transfer is linear and proportional to the amount of protein loaded.
Figure 4. Signal response from No-Stain labeling of proteins on membranes is linear. A Bolt 4-12% Bis-Tris Plus mini gel was loaded with HeLa lysate ranging from 1 to 50 µg, and PageRuler Unstained Protein Ladder in lane 1. After electrophoresis using MES running buffer, proteins were transferred onto a PVDF membrane using the Invitrogen PowerBlotter and PowerBlotter Select Stacks (10 mins). The PVDF membrane was washed twice for 2 minutes with 20 mL of ultra-pure water on a rotating platform. The No-Stain labeling reaction was initiated by the addition of 10 mL of the No-Stain Labeling Solution to the dish containing the PVDF membrane. The labeling reaction was allowed to proceed for 10 minutes on a rotating platform. The membrane was then washed 3 times for 2 minutes with 20 mL of ultra-pure water on a rotating platform. The image was captured using an iBright Imager with the No-Stain Membrane epi setting (455—485 nm excitation and 565—615 emission).
Quantitative gel staining
Figure 5 illustrates the linearity of signal to protein quantity loaded when using the No-Stain Protein Labeling Reagent to label proteins in gels.
Figure 5. Quantitative gel staining. A Bolt 4—12% Bis-Tris Plus mini gel was loaded with HeLa lysate concentrations ranging from 2.5 to 80 µg and electrophoresed with MES running buffer. After electrophoresis, the proteins in the gel were labeled following the No-Stain Protein Labeling Reagent protocol for labeling proteins in a gel, and the gel was imaged using an iBright imager with the transilluminator for excitation (490—520 nm) and the 565—615 nm emission filter.
Signal Intensity of No-Stain Labeled Proteins increases with incubation time
No-Stain Protein LabelingReagent sensitivity can be fine-tuned to improve the detection of low-abundance proteins. Increasing the incubation time of gels or blots with the No-Stain Reagent produces a linear increase in signal intensity. Alternatively, when time is limited, doubling the concentration of No-Stain Protein Labeling Reagent, produces a similar result with shorter labeling time. Exposure times can be easily set and adjusted to accommodate experimental conditions with an iBright Imaging System, extending the method detection capabilities to a broader range of protein expression levels.
Figure 6: Prolonging the incubation time increases the sensitivity of No-Stain Labeling Reagent. Transferred PVDF (polyvinylidene fluoride) membranes, incubated with No-Stain Labeling Reagent for 10, 20, 30 and 40 minutes respectively, show increased signal intensity over time. Invitrogen NuPAGE 4-12% Bis-Tris gels were loaded with E.Coli lysate ranging from 40 to 1.25 μg and separated by electrophoresis using MES SDS running buffer. Proteins from the gels were transferred onto mini PVDF membranes using the Invitrogen iBlot 2 Gel Transfer Device with iBlot 2 Transfer Stacks (P0 protocol for 7 minutes). The PVDF membranes were quickly rinsed with 20 mL of ultrapure water and incubated with 10 mL of a working solution of No-Stain Protein Labeling Reagent on a rotating platform. Analysis images, without brightness and contrast adjustment, of the blots were collected using the Invitrogen iBright FL1500 Imaging System after 10, 20, 30, and 40 minutes after addition of the No-Stain Protein Labelling Reagent (exposure time 1.000 seconds). Longer exposure times will result in stronger signals allowing for improved detection of low-expressed proteins (A). A linear correlation between the normalized signal intensity plotted against the incubation time can be observed (B).
Doubling the concentration of No-Stain Protein Labeling Reagent produces stronger signals
Figure 7. Doubling the concentration of No-Stain Labeling Reagent increases the sensitivity of the detection. Invitrogen Bolt 4-12% Bis-Tris Plus gels were loaded with A431 lysate (20 μg to 20 ng serial dilutions) in Bolt LDS sample buffer and separated by electrophoresis using MES SDS running buffer. Proteins were labeled with 1x or 2x No-Stain Labeling Reagent directly on the gel. 1X No-Stain Labeling Reagent was prepared according to the No-Stain Protein Labeling Reagent standard protocol. 2X No-Stain Protein Labeling Reagent was prepared by doubling the concentration of No-Stain Activator (40 μL) and No-Stain Derivatizer (40 μL). Analysis images, without brightness and contrast adjustment, of gels (A) and membranes (B) were collected using the Invitrogen iBright FL1500 Imaging System (exposure time 1.000 seconds).
Immunoblotting of No-Stain Labeled Gels
No-Stain Protein Labeling Reagent versatility extends to protein transfer and immunoblotting. Protein labeling with No-Stain Reagent does not affect protein transfer efficiency and is compatible with downstream western blotting, as shown in Figure 8.
Figure 8. No-Stain Labeling Reagent is compatible with protein transfer and immunoblotting. Invitrogen Bolt 4-12% Bis-Tris Plus gels were loaded with A431 lysate (20 μg to 20 ng serial dilutions) in Bolt LDS sample buffer and separated by electrophoresis using MES SDS running buffer. The gels were incubated with No-Stain Protein Labeling Reagent for 10 minutes on a rotating platform. No-Stain Labeled gels were transferred to PVDF membranes using the Invitrogen iBlot 2 Gel Transfer Device with iBlot 2 Transfer Stacks (P0 protocol for 7 minutes). PVDF membranes were then probed with specific primary antibodies and proteins were detected using secondary antibodies labeled with the Alexa fluor 800 dye. Analysis images, without brightness and contrast adjustment, were collected using the Invitrogen iBright FL1500 Imaging System (exposure time 1.000 seconds).
As Sensitive as Coomassie staining in a fraction of the time
No-Stain Protein Labeling Reagent provides a fast sensitive way to visualize proteins on gels and membranes without lengthy destaining steps and exposure to methanol.
Figure 9. The sensitivity of No-Stain Labeling Reagent is comparable to Coomassie staining. Invitrogen Bolt 4-12% Bis-Tris Plus gels were loaded with A431 lysate (20 μg to 20 ng serial dilutions) in Bolt LDS sample buffer and separated by electrophoresis using MES SDS running buffer. Protein bands were either labeled with No-Stain Protein Labeling Reagent or stained with Coomassie. Analysis images, without brightness and contrast adjustment, were collected using the Invitrogen iBright FL1500 Imaging System (exposure time 1.000 seconds). Additionally, the exposure time of the No-Stain image could be increased to allow for improved detection of even lower lysate loads compared to Coomassie.
The No-Stain Protein Labeling Reagent is compatible with chemiluminescent and fluorescent detection. Therefore, you can use your current chemiluminescent secondary antibody enzyme conjugates. When designing experiments using fluorescent antibody conjugates, it is important to choose fluorophores that do not overlap with the excitation and emission spectra of the No-Stain label, which, when covalently bound to lysine residues emits light at ~590 nm when excited by an ~488 nm light source. The choice of one fluorophore over another will depend on the filter set of your fluorescence imager.
You can compare the excitation and emission spectra of your secondary antibody's fluorescent conjugates to that of the No-Stain label to assess compatibility with your current antibody conjugates. The excitation and emission spectra of the covalently linked No-Stain label is shown in Figure 6.
Compare spectra of your antibody conjugates using our Spectra Viewer
Table 1. Compatible fluorescent secondary antibodies when using the No-Stain Protein Labeling Reagent with the iBright FL1500 Imaging System.
| Alexa Fluor Conjugate | Excitation/Emission (nm) | Alexa Fluor Secondary Antibodies |
|---|---|---|
| Alexa Fluor 635 | 621/639 | See antibodies |
| Alexa Fluor 647 Alexa Fluor Plus 647 | 650/665 | See antibodies |
| Alexa Fluor 660 | 660/689 | See antibodies |
| Alexa Fluor 680 Alexa Fluor Plus 680 | 679/702 | See antibodies |
| Alexa Fluor 700 | 702/724 | See antibodies |
| Alexa Fluor 750 | 749/775 | See antibodies |
| Alexa Fluor 790 | 784/814 | See antibodies |
| Alexa Fluor Plus 800 | 777/794 | See antibodies |
Figure 10. Excitation and emission spectra of the covalently linked No-Stain label. The excitation maximum of the the covalently linked No-Stain label is ~488 nm and its emission maximum is 590 nm. The spectra show that the signal from the No-Stain label can be imaged using a UV or fluorescent light source and the signal can be captured with a wide range of emission filters.
Table 1 is a partial list of fluorophores that are compatible with the covalently linked No-Stain label when imaged using the iBright FL1500 Imaging System. Any of the conjugates can be used individually with No-Stain labeling and the iBright imager. If multiplexing using two fluorophores and the No-Stain Protein Labeling Reagent, select one fluorophore from the 600 nm range and the second fluorophore from the 700 nm range so that each can be imaged by a distinct iBright emission filter.
Table 1. Compatible fluorescent secondary antibodies when using the No-Stain Protein Labeling Reagent with the iBright FL1500 Imaging System.
| Number of reactions | A standard kit will label 40 mini gel-sized membranes or 40 mini gels, or a combination of the two |
| Reaction time | 10 minutes |
| Gel compatibility | Compatible with any gel type (precast or pour your own) or gel chemistry (Bis-Tris, Tris-glycine, Tris-acetate, Tricine) |
| Membrane compatibility | PVDF, nitrocellulose |
| Immunodetection reagent compatibility | Compatible with all downstream immunodetection steps with chemiluminescence or fluorescence-based detection |
| Excitation and emission maxima | Excitation: ~488 nm (epi blue light) Emission: 590 nm |
| Detection sensitivity | 20 ng per band |
| Linear range | 1–80 μg total protein load per lane |
| Imager requirements | Compatible with a wide range of imagers that have UV or fluorescence light sources, for example, the iBright FL1500 Imaging System |
| How does it work? | The No-Stain Protein Labeling Reagent forms covalent bonds with a portion of the lysine amino acid side chains on all protein samples |
| Kit Component and storage conditions | No-Stain Activator (800 µL; storage -20⁰C); No-Stain Derivatizer (800 µL; storage -20⁰C); No-Stain Labeling Buffer, 20X (2 x 20 mL each; storage 15—30⁰C) |
| Shelf-life | At least 2 years |
No-Stain protocol for labeling protein on gels or transferred blots
Figure 1: No-Stain Protein Labeling Reagent Protocol
Preparation of No-Stain Protein Labeling Reagent
Mix the reagents needed to prepare the appropriate volume of No-Stain Protein Labeling Reagent as indicated in the table.
| No-Stain Protein Labeling Reagent Volume, 1X | No-Stain Labeling buffer, 20X | Ultrapure water | No-Stain Activator | No-Stain Derivatizer | |
|---|---|---|---|---|---|
| Mini gel | 20 mL | 1 mL | 19 mL | 20 µL | 20 µL |
| Mini membrane | 10 mL | 0.5 mL | 9.5 mL | 20 µL | 20 µL |
| Midi membrane | 20 mL | 1 mL | 19 mL | 40 µL | 40 µL |
In western blotting, variable results can be caused by unequal protein sample concentration, inconsistent sample loading onto the gel, and/or transfer variation during electroblotting. Protein normalization is used to correct for this variability and is a critical step in obtaining reliable and reproducible quantitative western blot data. Methods for protein normalization include use of internal housekeeping protein controls such as GAPDH, β-tubulin, β-actin, or cyclophilin B, exogenous loading controls, or total protein normalization.
In recent years, journal editors and reviewers have been requesting adoption of methods that improve the accuracy and reproducibility of quantitative western blot data. Many leading journals have developed guidelines for submitting western blotting research and select quotes from those guidelines are highlighted below.
- “For quantitative comparisons, appropriate reagents, controls and imaging methods with linear signal ranges should be used” – Nature
- “Record how data were obtained, whether signal intensity was linear with antigen loading, and how protein loading was normalized” – Journal of Biological Chemistry
- “Normalize signal intensity to total protein loading (assessed by staining membranes for total protein) whenever possible” – Journal of Biological Chemistry
- “House-keeping proteins should not be used for normalization without evidence that manipulations do not affect expression” – Journal of Biological Chemistry
The use of housekeeping proteins has its drawbacks as the expression of housekeeping gene proteins can vary with experimental conditions, and they can often have oversaturated western blotting signals.
Total protein normalization using the No-Stain Protein Labeling Reagent avoids the variability and inaccuracy of using housekeeping proteins and avoids the time, effort, and cost to detect the housekeeping gene protein using immunoblotting, which may involve stripping of the blot and re-probing.
An accurate loading control should display a linear relationship between signal intensity and sample load under all experimental conditions. The signal intensity obtained from labeling of total proteins on a membrane with No-Stain reagent ensures a linear relationship between signal intensity and sample load (Figure 2) in all experimental conditions. Therefore, the No-Stain reagent enables the use of total protein as an ideal loading control for quantitative western blotting applications.
Comparison of linearity of the No-Stain reagent to housekeeping proteins
The graph in Figure 2 below shows the linear signal response versus the amount of protein loaded per well using the No-Stain Protein Labeling Reagent for total protein normalization. Signals from the housekeeping proteins appear to be saturating at higher lysate loads and would provide less accurate normalization of results.
Figure 2. Total protein normalization using the No-Stain Protein Labeling Reagent: Bolt 4-12% Bis-Tris Plus gels were loaded with HeLa Lysate ranging from 10 to 50 µg and electrophoresed using MES running buffer. Proteins from the gels were transferred onto PVDF membranes using the Invitrogen iBlot 2 Gel Transfer Device with iBlot 2 Transfer Stacks, PVDF, mini (P0 protocol for 7 minutes). The PVDF membranes were washed twice for 2 minutes with 20 mL of ultra-pure water on a rotating platform, whereupon they were labeled with 10 mL of No-Stain labeling solution on a rotating platform for 10 minutes. The membranes were then washed 3 times for 2 minutes with 20 mL of ultra-pure water on a rotating platform, followed by immunoblotting for β-actin (Cat. no. AM4302), GAPDH (Cat. no. 398600), and α-tubulin (Cat. no. 138000) followed by goat anti-mouse Alexa Fluor Plus 680 (Cat. no. A21058). The blot was imaged using the iBright Imager. The iBright software was used to quantitate the total protein signal in the lanes. The linear regression value of the plotted data for the entire load range using the No-Stain Protein Labeling Reagent was determined (R2 = 0.9990), whereas the R2 values for β-actin, GAPDH, and α-tubulin were 0.8851, 0.9438, and 0.8332, respectively.
Figure 3 below illustrates normalization of a target protein using the No-Stain Protein Labeling Reagent with a nitrocellulose membrane.
Figure 3. Quantitative western blot analysis using the No-Stain Protein Labeling Reagent and an iBright Imaging System. A Novex 4-12% Tris-Glycine gel, WedgeWell format, was loaded and electrophoresed with lysates from HeLa cells expressing RB1, at total protein loads ranging from 0.6 to 10 µg. Proteins from the gel were transferred to a nitrocellulose membrane using the iBlot 2 Dry Blotting System. The nitrocellulose membrane was labeled using the No-Stain Protein Labeling Reagent for 10 minutes and the labeled membrane was imaged using the iBright imager. The same No-Stain labeled membrane was used to probe for RB1 with a specific antibody labeled with Alexa Fluor 645 dye. The iBright normalization software was used to quantify the total protein signal in lanes loaded with HeLa lysate loads ranging from 0.6 to 10 µg and signal intensities from RB1 immunodetection bands. The signal intensity from the total protein load and RB1 were plotted.
Labeling proteins in gels using the No-Stain Protein Labeling Reagent
The No-Stain Protein Labeling Reagent forms covalent bonds with a portion of the lysine amino acid side chains present in all proteins in a gel within 10 minutes.
- A great alternative to traditional gel staining reagents—No destaining is required and allows for accurate normalization over a wide range of protein lysate loads (1—80 µg)
- Sensitive—Lower limit of detection of 20 ng per band
- Specific—Forms bonds only with the lysine side chains of proteins. Free label does not fluoresce, thereby enabling a superior signal-to-noise ratio
- Flexible—Compatible with any gel type; no need to change your gels to get stain-free convenience
Figure 4 below shows labeling of protein on a blot with No-Stain Protein Labeling Reagent after transfer is linear and proportional to the amount of protein loaded.
Figure 4. Signal response from No-Stain labeling of proteins on membranes is linear. A Bolt 4-12% Bis-Tris Plus mini gel was loaded with HeLa lysate ranging from 1 to 50 µg, and PageRuler Unstained Protein Ladder in lane 1. After electrophoresis using MES running buffer, proteins were transferred onto a PVDF membrane using the Invitrogen PowerBlotter and PowerBlotter Select Stacks (10 mins). The PVDF membrane was washed twice for 2 minutes with 20 mL of ultra-pure water on a rotating platform. The No-Stain labeling reaction was initiated by the addition of 10 mL of the No-Stain Labeling Solution to the dish containing the PVDF membrane. The labeling reaction was allowed to proceed for 10 minutes on a rotating platform. The membrane was then washed 3 times for 2 minutes with 20 mL of ultra-pure water on a rotating platform. The image was captured using an iBright Imager with the No-Stain Membrane epi setting (455—485 nm excitation and 565—615 emission).
Quantitative gel staining
Figure 5 illustrates the linearity of signal to protein quantity loaded when using the No-Stain Protein Labeling Reagent to label proteins in gels.
Figure 5. Quantitative gel staining. A Bolt 4—12% Bis-Tris Plus mini gel was loaded with HeLa lysate concentrations ranging from 2.5 to 80 µg and electrophoresed with MES running buffer. After electrophoresis, the proteins in the gel were labeled following the No-Stain Protein Labeling Reagent protocol for labeling proteins in a gel, and the gel was imaged using an iBright imager with the transilluminator for excitation (490—520 nm) and the 565—615 nm emission filter.
Signal Intensity of No-Stain Labeled Proteins increases with incubation time
No-Stain Protein LabelingReagent sensitivity can be fine-tuned to improve the detection of low-abundance proteins. Increasing the incubation time of gels or blots with the No-Stain Reagent produces a linear increase in signal intensity. Alternatively, when time is limited, doubling the concentration of No-Stain Protein Labeling Reagent, produces a similar result with shorter labeling time. Exposure times can be easily set and adjusted to accommodate experimental conditions with an iBright Imaging System, extending the method detection capabilities to a broader range of protein expression levels.
Figure 6: Prolonging the incubation time increases the sensitivity of No-Stain Labeling Reagent. Transferred PVDF (polyvinylidene fluoride) membranes, incubated with No-Stain Labeling Reagent for 10, 20, 30 and 40 minutes respectively, show increased signal intensity over time. Invitrogen NuPAGE 4-12% Bis-Tris gels were loaded with E.Coli lysate ranging from 40 to 1.25 μg and separated by electrophoresis using MES SDS running buffer. Proteins from the gels were transferred onto mini PVDF membranes using the Invitrogen iBlot 2 Gel Transfer Device with iBlot 2 Transfer Stacks (P0 protocol for 7 minutes). The PVDF membranes were quickly rinsed with 20 mL of ultrapure water and incubated with 10 mL of a working solution of No-Stain Protein Labeling Reagent on a rotating platform. Analysis images, without brightness and contrast adjustment, of the blots were collected using the Invitrogen iBright FL1500 Imaging System after 10, 20, 30, and 40 minutes after addition of the No-Stain Protein Labelling Reagent (exposure time 1.000 seconds). Longer exposure times will result in stronger signals allowing for improved detection of low-expressed proteins (A). A linear correlation between the normalized signal intensity plotted against the incubation time can be observed (B).
Doubling the concentration of No-Stain Protein Labeling Reagent produces stronger signals
Figure 7. Doubling the concentration of No-Stain Labeling Reagent increases the sensitivity of the detection. Invitrogen Bolt 4-12% Bis-Tris Plus gels were loaded with A431 lysate (20 μg to 20 ng serial dilutions) in Bolt LDS sample buffer and separated by electrophoresis using MES SDS running buffer. Proteins were labeled with 1x or 2x No-Stain Labeling Reagent directly on the gel. 1X No-Stain Labeling Reagent was prepared according to the No-Stain Protein Labeling Reagent standard protocol. 2X No-Stain Protein Labeling Reagent was prepared by doubling the concentration of No-Stain Activator (40 μL) and No-Stain Derivatizer (40 μL). Analysis images, without brightness and contrast adjustment, of gels (A) and membranes (B) were collected using the Invitrogen iBright FL1500 Imaging System (exposure time 1.000 seconds).
Immunoblotting of No-Stain Labeled Gels
No-Stain Protein Labeling Reagent versatility extends to protein transfer and immunoblotting. Protein labeling with No-Stain Reagent does not affect protein transfer efficiency and is compatible with downstream western blotting, as shown in Figure 8.
Figure 8. No-Stain Labeling Reagent is compatible with protein transfer and immunoblotting. Invitrogen Bolt 4-12% Bis-Tris Plus gels were loaded with A431 lysate (20 μg to 20 ng serial dilutions) in Bolt LDS sample buffer and separated by electrophoresis using MES SDS running buffer. The gels were incubated with No-Stain Protein Labeling Reagent for 10 minutes on a rotating platform. No-Stain Labeled gels were transferred to PVDF membranes using the Invitrogen iBlot 2 Gel Transfer Device with iBlot 2 Transfer Stacks (P0 protocol for 7 minutes). PVDF membranes were then probed with specific primary antibodies and proteins were detected using secondary antibodies labeled with the Alexa fluor 800 dye. Analysis images, without brightness and contrast adjustment, were collected using the Invitrogen iBright FL1500 Imaging System (exposure time 1.000 seconds).
As Sensitive as Coomassie staining in a fraction of the time
No-Stain Protein Labeling Reagent provides a fast sensitive way to visualize proteins on gels and membranes without lengthy destaining steps and exposure to methanol.
Figure 9. The sensitivity of No-Stain Labeling Reagent is comparable to Coomassie staining. Invitrogen Bolt 4-12% Bis-Tris Plus gels were loaded with A431 lysate (20 μg to 20 ng serial dilutions) in Bolt LDS sample buffer and separated by electrophoresis using MES SDS running buffer. Protein bands were either labeled with No-Stain Protein Labeling Reagent or stained with Coomassie. Analysis images, without brightness and contrast adjustment, were collected using the Invitrogen iBright FL1500 Imaging System (exposure time 1.000 seconds). Additionally, the exposure time of the No-Stain image could be increased to allow for improved detection of even lower lysate loads compared to Coomassie.
The No-Stain Protein Labeling Reagent is compatible with chemiluminescent and fluorescent detection. Therefore, you can use your current chemiluminescent secondary antibody enzyme conjugates. When designing experiments using fluorescent antibody conjugates, it is important to choose fluorophores that do not overlap with the excitation and emission spectra of the No-Stain label, which, when covalently bound to lysine residues emits light at ~590 nm when excited by an ~488 nm light source. The choice of one fluorophore over another will depend on the filter set of your fluorescence imager.
You can compare the excitation and emission spectra of your secondary antibody's fluorescent conjugates to that of the No-Stain label to assess compatibility with your current antibody conjugates. The excitation and emission spectra of the covalently linked No-Stain label is shown in Figure 6.
Compare spectra of your antibody conjugates using our Spectra Viewer
Table 1. Compatible fluorescent secondary antibodies when using the No-Stain Protein Labeling Reagent with the iBright FL1500 Imaging System.
| Alexa Fluor Conjugate | Excitation/Emission (nm) | Alexa Fluor Secondary Antibodies |
|---|---|---|
| Alexa Fluor 635 | 621/639 | See antibodies |
| Alexa Fluor 647 Alexa Fluor Plus 647 | 650/665 | See antibodies |
| Alexa Fluor 660 | 660/689 | See antibodies |
| Alexa Fluor 680 Alexa Fluor Plus 680 | 679/702 | See antibodies |
| Alexa Fluor 700 | 702/724 | See antibodies |
| Alexa Fluor 750 | 749/775 | See antibodies |
| Alexa Fluor 790 | 784/814 | See antibodies |
| Alexa Fluor Plus 800 | 777/794 | See antibodies |
Figure 10. Excitation and emission spectra of the covalently linked No-Stain label. The excitation maximum of the the covalently linked No-Stain label is ~488 nm and its emission maximum is 590 nm. The spectra show that the signal from the No-Stain label can be imaged using a UV or fluorescent light source and the signal can be captured with a wide range of emission filters.
Table 1 is a partial list of fluorophores that are compatible with the covalently linked No-Stain label when imaged using the iBright FL1500 Imaging System. Any of the conjugates can be used individually with No-Stain labeling and the iBright imager. If multiplexing using two fluorophores and the No-Stain Protein Labeling Reagent, select one fluorophore from the 600 nm range and the second fluorophore from the 700 nm range so that each can be imaged by a distinct iBright emission filter.
Table 1. Compatible fluorescent secondary antibodies when using the No-Stain Protein Labeling Reagent with the iBright FL1500 Imaging System.
References
- Paramanantham, A., Asfiya, R., Das, S., & Mccully, G. (2023). No-stain protein labeling as a potential normalization marker for small extracellular vesicle proteins. Preparative Biochemistry & Biotechnology, 0(0), 1–11.
- Lazcano, P., Schmidtke, M. W., Onu, C. J., & Greenberg, M. L. (2022). Phosphatidic acid inhibits inositol synthesis by inducing nuclear translocation of kinase IP6K1 and repression of myo -inositol-3-P synthase. Journal of Biological Chemistry, 298(9), 102363.
- Taylor, S. C., Murai, L. K. R., Crobeddu, B., & Plante, I. (2022). A critical path to producing high quality , reproducible data from quantitative western blot experiments. Scientific Reports.
- Matassa, D. S., Criscuolo, D., Avolio, R., Agliarulo, I., Sarnataro, D., Pacelli, C., Scrima, R., Colamatteo, A., Matarese, G., & Capitanio, N. (2022). Regulation of mitochondrial complex III activity and assembly by TRAP1 in cancer cells. Cancer Cell International, 22(402), 1–18.
Resources
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