Invitrogen No-Stain Protein Labeling Reagent for quantifying western blot data

Total protein normalization

Total protein normalization is a useful method for obtaining accurate, quantitative western blotting data, as housekeeping proteins can often be affected by experimental conditions. The Invitrogen No-Stain Protein Labeling Reagent is a fast, easy to use, covalent protein labeling reagent, applied to a membrane after gel 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 gel stain for visualization of proteins in a gel.

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No-Stain Protein Labeling Reagent

  • Flexible use labeling format—Use with any protein gel of choice to perform total protein labeling of the membrane post-transfer. Or use it as a fast protein stain after electrophoresis of gels you do not intend to transfer.
  • Easy to use protocol—Mix and incubate with the transferred membrane. The reaction time is 10 minutes. Gel staining is also a 10 minute reaction.
  • Flexible visualization—Use a wide-range of imagers with UV, Green LED or Fluorescence (~488 mm) light source, including the iBright FL1500 Imaging System, to capture an image.
  • Accurate total protein normalization—Designed to provide accurate total protein normalization over a broad linear range for protein detection: 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.
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 proteins
Kit Component and storage conditions No-Stain Activator (800 µL; storage -20⁰ C);
No-Stain Derivatizer (800 µL; storage -20⁰ C);
No-Stain Labeling Buffer, 20X (2 x 20 mL each; storage 15-30⁰ C)
Shelf-life 1 year from date of manufacture

No-Stain protocol for labeling protein on transferred blots

Invitrogen No-Stain Protein Labeling Reagent protocol
Figure 1. Labeling a protein blot with No-Stain Protein Labeling Reagent after transfer.

Immediately after transferring gel to nitrocellulose or PVDF:
Wash the transferred membrane with 20 mL water for 2 minutes, on a rotating platform at ~60 rpm. Repeat the wash for a total of 2 washes.

For No-Stain Protein Labeling Reagent preparation and labeling of proteins on a membrane:
  1. Prepare 10 mL of 1X No-Stain Labeling Buffer by diluting 0.5 mL No-Stain Labeling Buffer, 20X with 9.5 mL water.
  2. Add 20 µL of No-Stain Activator to 1X No-Stain Labeling Buffer.
  3. Add 20 µL of No-Stain Derivatizer to the mixture in step 2.
  4. Pour the prepared No-Stain Protein Labeling Solution onto the washed blot and rotate for 10 minutes.
Wash the blot after the protein labeling step with 20 mL water for 2 minutes, on a rotating platform. Repeat the wash for a total of 3 washes.
Image blot or place in blocking buffer to process the western blot.

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.

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.

Quantitative western blot analysis using Invitrogen No-Stain Protein Labeling Reagent
Total protein normalization with the No-Stain Protein Labeling Reagent - Figure 2 B

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 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.

Linear staining of Invitrogen No-Stain Protein Labeling Reagent on PVDF membranes
Linear staining of Invitrogen No-Stain Protein Labeling Reagent with cell lysate quantities from 1-50 µg.

Figure 3. 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 minutes). 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).

Figure 4 below illustrates normalization of a target protein using the No-Stain Protein Labeling Reagent with a nitrocellulose membrane.

Quantitative western blotting analysis with No-Stain Protein Labeling Reagent and iBright Imaging System.
Quantitative western blotting analysis with No-Stain Protein Labeling Reagent and iBright Imaging System

Figure 4. 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 prsent 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.

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.

Quantitative gel staining using Invitrogen No-Stain Protein Labeling Reagent
Quantitative gel staining using Invitrogen No-Stain Protein Labeling Reagent

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.

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.

Excitation and Emission spectra of labeled proteins with Invitrogen No-Stain Protein Labeling Reagent
 Click image to enlarge

Figure 6. 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.

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
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 proteins
Kit Component and storage conditions No-Stain Activator (800 µL; storage -20⁰ C);
No-Stain Derivatizer (800 µL; storage -20⁰ C);
No-Stain Labeling Buffer, 20X (2 x 20 mL each; storage 15-30⁰ C)
Shelf-life 1 year from date of manufacture

No-Stain protocol for labeling protein on transferred blots

Invitrogen No-Stain Protein Labeling Reagent protocol
Figure 1. Labeling a protein blot with No-Stain Protein Labeling Reagent after transfer.

Immediately after transferring gel to nitrocellulose or PVDF:
Wash the transferred membrane with 20 mL water for 2 minutes, on a rotating platform at ~60 rpm. Repeat the wash for a total of 2 washes.

For No-Stain Protein Labeling Reagent preparation and labeling of proteins on a membrane:
  1. Prepare 10 mL of 1X No-Stain Labeling Buffer by diluting 0.5 mL No-Stain Labeling Buffer, 20X with 9.5 mL water.
  2. Add 20 µL of No-Stain Activator to 1X No-Stain Labeling Buffer.
  3. Add 20 µL of No-Stain Derivatizer to the mixture in step 2.
  4. Pour the prepared No-Stain Protein Labeling Solution onto the washed blot and rotate for 10 minutes.
Wash the blot after the protein labeling step with 20 mL water for 2 minutes, on a rotating platform. Repeat the wash for a total of 3 washes.
Image blot or place in blocking buffer to process the western blot.

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.

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.

Quantitative western blot analysis using Invitrogen No-Stain Protein Labeling Reagent
Total protein normalization with the No-Stain Protein Labeling Reagent - Figure 2 B

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 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.

Linear staining of Invitrogen No-Stain Protein Labeling Reagent on PVDF membranes
Linear staining of Invitrogen No-Stain Protein Labeling Reagent with cell lysate quantities from 1-50 µg.

Figure 3. 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 minutes). 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).

Figure 4 below illustrates normalization of a target protein using the No-Stain Protein Labeling Reagent with a nitrocellulose membrane.

Quantitative western blotting analysis with No-Stain Protein Labeling Reagent and iBright Imaging System.
Quantitative western blotting analysis with No-Stain Protein Labeling Reagent and iBright Imaging System

Figure 4. 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 prsent 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.

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.

Quantitative gel staining using Invitrogen No-Stain Protein Labeling Reagent
Quantitative gel staining using Invitrogen No-Stain Protein Labeling Reagent

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.

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.

Excitation and Emission spectra of labeled proteins with Invitrogen No-Stain Protein Labeling Reagent
 Click image to enlarge

Figure 6. 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.

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