TECHNICAL NOTE pubs.acs.org/jpr
Coomassie Staining as Loading Control in Western Blot Analysis Charlotte Welinder and Lars Ekblad* Department of Oncology, Clinical Sciences, Lund University, Sweden ABSTRACT: In Western blotting, immunodetection of housekeeping proteins is routinely performed to detect differences in electrophoresis loading. The present work describes a much faster and simpler protein staining method, which is compatible with ordinary blocking conditions. In addition, the method can be used after immunodetection with superior linearity compared to ordinary staining methods. After immunoblotting and staining, protein bands can be further identified using peptide mass fingerprinting. KEYWORDS: Western blot, loading control, Coomassie, MALDITOF, PVDF, GAPDH, peptide mass fingerprinting
’ INTRODUCTION Western blotting is a powerful and widely used technique for specifically measuring the content of single proteins in complex biological mixtures. Two factors that have to be controlled to ensure reliable comparisons of proteins amounts in different samples are the total amount of protein loaded to each well in the electrophoretic separation and the efficiency of transfer in the blotting procedure. For intrasample comparisons, loading exactness is less crucial but variations in transfer efficiency over the blotting membrane can cause spurious results. To address these possible sources of error, it has become popular to use antibodies directed against housekeeping proteins that are (often only supposedly) constantly expressed, as loading controls. Commonly used proteins for this purpose are, for example, actin, β-tubulin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). However, several problems are associated with this procedure: the expression of the control protein might in fact differ between the samples, variations in transfer efficiency are not effectively detected, for low abundant proteins the gel might be overloaded with regard to the control protein,1 the analyzed protein might overlap in apparent molecular weight with the control protein, and last but not least, the extra antibody detection step is both time-consuming and expensive. In the present work, we show that it is possible to use a conventional Coomassie staining procedure after the immunodetection of proteins blotted onto polyvinylidene fluoride (PVDF) membranes to control the total protein load and the blotting efficiency. We also show that the method has superior linearity compared with antibody detection of housekeeping proteins and that it is also compatible with mass spectrometric protein identification.
protein, we lysed 107 LU-HNSCC-4 cells2 in 500 μL of 1% Triton X-100, 150 mmol/L NaCl, Complete protease inhibitor cocktail, 1 tablet per 50 mL buffer, (Roche Applied Science, Basel, Switzerland), 50 mmol/L Tris-HCl, pH 7.4. The protein concentration of the lysate was measured by the micro BCA protein assay (Thermo Scientific, Rockford, IL) using bovine serum albumin as standard. The lysate was diluted in NuPAGE LDS sample buffer (Invitrogen, Carlsbad, CA) with 50 mmol/L dithiotreitol (DTT) to a protein concentration of 1.67 μg/μL and incubated at 60 °C for 10 min. This mixture was diluted with the same buffer to different concentrations. Equal volumes of each dilution were loaded on 4-12% NuPAGE, Bis-Tris, 1 mm thick gels with 15 wells (Invitrogen). The electrophoresis was run in MOPS buffer at 180 V for 1 h and the proteins were then transferred to Immobilon-P PVDF membrane (Millipore, Billerica, MA) in 25 mmol/L Tris and 192 mM glycine at 350 mA for 1 h. The membrane was blocked in 5% nonfat dry milk (alternatively with 0.4% gelatin or 3% BSA) in 0.2% Tween-20 (or alternatively with other concentrations as stated), 150 mmol/ L NaCl and 20 mmol/L Tris, pH 7.5 (TTBS) for 1 h and incubated with anti-GAPDH antibody (cat. no 2118, Cell Signaling Technology, Danvers, MA), diluted 1:1000, followed by two washes in TTBS, 5 min each. Then the membrane was incubated with HRP-linked antirabbit IgG (cat. no 7074, Cell Signaling Technology), diluted 1:3000, both in blocking solution. The membrane was washed four times, 10 min each, in TTBS and antibody binding was detected with the ECLplus reagent chemiluminescence detection system (GE Healthcare, Waukesha, WI). The staining intensity was determined using a FluorChem FC2 with AlphaView software (Cell Biosciences, Santa Clara, CA). Coomassie Staining
’ EXPERIMENTAL SECTION
After immunodetection, the membrane was washed twice with TTBS and then stained with 0.1% Coomassie R-350 (GE
Electrophoresis and Blotting
To assess the linearity of the protein staining method in comparison with that of antibody detection of a housekeeping r 2010 American Chemical Society
Received: September 3, 2010 Published: December 27, 2010 1416
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TECHNICAL NOTE
Figure 1. Linearity of Coomassie protein stain and GAPDH immunostaining. (A) A dilution series of a cell lysate was detected with anti-GAPDH and (B) then the proteins were stained with Coomassie R-350. Quantification of the GAPDH bands and the protein lanes were performed using the AlphaView software. (C) Relative densities, in comparison to the lowest protein load, of GAPDH (open triangles) and protein staining (filled circles) were plotted against the protein amount per well ( standard deviation. Comparison of different blocking conditions. Membranes were blocked with TBS containing 5% nonfat dry milk with addition of 0% (diamonds), 0.05% (triangles) or 0.2% (circles) Tween-20. (D) Measurements were not background corrected.
Healthcare) in methanol/water, 1:1, for 1 min, destained for 20 min in acetic acid/ethanol/water, 1:5:4, washed with water and air-dried. The dry membrane was scanned in a flat-bed scanner at 600 dpi and the staining density for each complete lane was analyzed in the AlphaView software with an area outside the protein lanes defining the background. Peptide Mass Fingerprinting
The protein bands were cut out with a scalpel blade from the air-dried membrane and treated essentially as previously described3 but with certain modifications. Each membrane piece was wet in 99.5% ethanol for 5 s and then washed once in 0.5 mL water, 30 min, and four times in 0.5 mL 40% acetonitrile (ACN), 25 mmol/L NH4HCO3 before reduction in 30 μL 10 mmol/L DTT, 25 mmol/L NH4HCO3 for 30 min at 56 °C, followed by alkylation with 30 μL 55 mmol/L iodoacetamide, 25 mmol/L NH4HCO3 for 30 min in darkness and one wash in 0.5 mL water, 30 min. The membrane pieces were blocked with 0.5% polyvinylpyrrolidone (PVP40, Sigma-Aldrich) in 100 mmol/L acetic acid, 30 min at 37 °C and then washed five times in 0.5 mL water and five times in 50 mmol/L NH4HCO3 before digestion with 10 μL 20 ng/μL sequencing-grade trypsin (Promega, Madison, WI) in 25 mmol/L NH4HCO3 at 37 °C, overnight. The digestion was terminated by the addition of 20 μL 2% trifluoroacetic acid
(TFA) and the membrane dried in a SpeedVac vacuum centrifuge. The resulting peptides were dissolved in 1 μL 50% ACN and 0.1% TFA in water and spotted directly onto the sample target (Anchorchip target, Bruker Daltonik GmbH, Bremen, Germany) where 0.7 μL of matrix, 2,5-dihydroxybenzoic acid (10 mg/mL in 30% ACN) had been added. Mass spectra of positively charged ions were recorded on a Bruker Reflex III instrument (Bruker Daltonik). The instrument was equipped with a delayed extraction ion source, utilizing a nitrogen laser at 337 nm and was operated in reflector mode at an accelerating voltage of 20 kV. A total of 160-210 single shot spectra were accumulated from each sample. The XMASS 5.0 and MS Biotools software packages provided by the manufactures were used for data processing. Known autoproteolysis products from the trypsin were used for internal calibration. For protein identification, protein sequences in the SwissProt database were searched using the Mascot Software (Matrix Science Ltd., London, U.K.). Parameters specified in the search were taxa, Homo sapiens; missed cleavages, 1; peptide mass tolerance, (0.1 Da; fixed modification; carbamidomethyl (C); variable modification, oxidized methionine. To search for contaminating peptides from antibodies or blocking proteins, searches were also performed with the taxa parameter set to mammalia. 1417
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TECHNICAL NOTE
Table 1. Peptide Mass Fingerprinting Identification sequence band no. 1
accession no.
protein
Mw matched coverage (kDa) peaks score (%)
GAPDH
P04406
36.2
14
150
Annexin A2
P07355
38.8
10
78
43 29
2
Actin,
P60709
42.7
15
151
47
3
Alpha-enolase
P06733
47.5
11
95
27
Elongation factor 1-alpha 1
P68104
50.5
11
57
26
Tubulin beta
P07437
50.1
13
135
30
P13639
96.2
15
86
20
cytoplasmic 1
4
chain 5
Elongation factor 2
Figure 2. Peptide mass fingerprinting. A cell lysate containing 10 μg total protein was stained with Coomassie R-350 after detection with anti-GAPDH. (A) Coomassie stained lane with the GAPDH immunodetected band overlaid and displayed in red color; (B) identical lane but without the GAPDH band. Protein band 1-5 were excised and identified by peptide mass fingerprinting (see Table 1).
’ RESULTS AND DISCUSSION To assess the linearity of the Coomassie staining versus the protein amount loaded per well, 2-25 μg total protein from a cell lysate was separated by electrophoresis, blotted to a PVDF membrane, detected with a GAPDH antibody (Figure 1A), and then stained with Coomassie R-350 (Figure 1B) essentially as described4 (see Experimental Section). The background staining contributed by the blocking proteins was negligible and, in essence, the resulting membrane was white with well resolved protein bands (Figure 1B). In addition to blocking with fat-free dry milk, we also tested gelatin and bovine serum albumin (BSA) with similar result (data not shown). The method is however not compatible with nitrocellulose membranes as this membrane type reacts with the Coomassie stain producing a dense background. After staining, the membrane was scanned and the total staining density of each lane analyzed with a quantification software. The resulting protein staining density was linear up to 20 μg protein per well (Figure 1C). This was in sharp contrast to the GAPDH staining (Figure 1A), which possibly was linear below 5 μg protein per well but certainly not above. We did not test different combinations of antibodies and detection systems in this study and can therefore not exclude that it is possible to find experimental conditions under which there is a better linearity for the GAPDH staining (or staining with antibodies against other proteins), but the results do show that, if not carefully controlled, the use of immunodetection of housekeeping proteins for loading control might produce erroneous results. It is clear that the use of the Coomassie protein staining in this set up offered a greatly improved linearity over immunodetection of GAPDH. A low background was also obtained if blocking was performed with lower Tween-20 concentrations (tested down to 0.025%). However, if Tween-20 was omitted from the blocking
buffer the background staining increased. This did not affect the linearity of the staining density, but a reliable background correction method has to be employed to obtain correct measurements (Figure 1D). Of course, in many cases blocking proteins can be omitted without affecting the blotting results. A second benefit with the protein staining method is that it also provides a quality control of the blotting process. As seen in the uppermost part of Figure 1B, the blotting produced a local artifact, which certainly would have affected immunodetection of proteins in this region. Using the protein staining method, it is easy to assess if poor protein transfer has affected the appearance of immunodetected band and to decide if the blot could be used for quantification or should be repeated. We also tested if it is possible to identify protein bands after both immunodetection and Coomassie staining using matrixassisted laser desorption ionization (MALDI)-time-of-flight (TOF) mass spectrometry. A protein band matching the GAPDH immunostaining (band 1) and four other, randomly chosen, protein bands (band 2-5) were excised and subjected to peptide mass fingerprinting (Figure 2, and Table 1). GAPDH was easily identified in the band detected by the GAPDH antibody. All the other identities matched the respective apparent molecular weights of the excised bands and were identified with high probability. There were no identities related to rabbit or goat IgG, or bovine milk proteins in any of the samples, which might be anticipated considering the antibody and protein blocking incubations. A more thorough comparison with other described methods for mass spectrometric identification of proteins in combination with Western blotting5-7 was not performed and it is likely that, for example, peptide recovery could be improved. Reversible Ponceau S protein staining prior to antibody detection has been used as an alternative loading control8,9 and for examining the transfer efficiency in Western blotting.10,11 Likewise, Coomassie staining and destaining and India ink have been employed to visualize proteins before antibody incubations.7,12 We think that the currently presented method has several attractive qualities compared to these methods: (1) the current Coomassie protein staining will under no circumstances affect the antibody binding as it is performed after all detection steps while the other methods are performed before antibody binding and thus might introduce artifacts affecting the immunodetection, (2) the protein bands can be easily matched with the immunostained image and excised for mass spectrometric identification, 1418
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Journal of Proteome Research and the Coomassie method (3) is more sensitive than the Ponceau method, (4) is less time-consuming than Coomassie staining and destaining and India ink staining, and (5) can be directly used in most common Western blotting work flows. The ease, low cost, reliability and compatibility with mass spectrometry mass fingerprinting of the presented Commassie based method supply strong motives for abandoning the routine use of immunodetection of so-called house-keeping proteins. Instead, we suggest that protein staining should be used for this purpose.
TECHNICAL NOTE
(12) Pryor, J. L.; Xu, W.; Hamilton, D. W. Immunodetection after complete destaining of coomassie blue-stained proteins on immobilonPVDF. Anal. Biochem. 1992, 202 (1), 100–4.
’ AUTHOR INFORMATION Corresponding Author
* Lars Ekblad Ph.D., Department of Oncology, Barngatan 2b, SE221 85 Lund, Sweden. E-mail:
[email protected]. Phone: þ46 46 17 85 33. Fax: þ46 46 14 73 27.
’ ACKNOWLEDGMENT This work was supported by Laryngfonden, the Crafoord Foundation, Magnus Bergvalls Foundation, Mrs. Berta Kamprad Foundation, and governmental funding of clinical research within the NHS. ’ REFERENCES (1) Dittmer, A.; Dittmer, J. Beta-actin is not a reliable loading control in Western blot analysis. Electrophoresis 2006, 27 (14), 2844–5. (2) Yamatodani, T.; Ekblad, L.; Kjellen, E.; Johnsson, A.; Mineta, H.; Wennerberg, J. Epidermal growth factor receptor status and persistent activation of Akt and p44/42 MAPK pathways correlate with the effect of cetuximab in head and neck and colon cancer cell lines. J. Cancer Res. Clin. Oncol. 2009, 135 (3), 395–402. (3) Welinder, C.; Jansson, B.; Fern€o, M.; Olsson, H.; Baldetorp, B. Expression of Helix pomatia lectin binding glycoproteins in women with breast cancer in relationship to their blood group phenotypes. J. Proteome Res. 2009, 8 (2), 782–7. (4) Matsudaira, P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J. Biol. Chem. 1987, 262 (21), 10035–8. (5) Lin, Y.; Li, Y.; Liu, Y.; Han, W.; He, Q.; Li, J.; Chen, P.; Wang, X.; Liang, S. Improvement of gel-separated protein identification by DMFassisted digestion and peptide recovery after electroblotting. Electrophoresis 2009, 30 (20), 3626–35. (6) Luque-Garcia, J. L.; Zhou, G.; Spellman, D. S.; Sun, T. T.; Neubert, T. A. Analysis of electroblotted proteins by mass spectrometry: protein identification after Western blotting. Mol. Cell. Proteomics 2008, 7 (2), 308–14. (7) Methogo, R. M.; Dufresne-Martin, G.; Leclerc, P.; Leduc, R.; Klarskov, K. Mass spectrometric peptide fingerprinting of proteins after Western blotting on polyvinylidene fluoride and enhanced chemiluminescence detection. J. Proteome Res. 2005, 4 (6), 2216–24. (8) Klein, D.; Kern, R. M.; Sokol, R. Z. A method for quantification and correction of proteins after transfer to immobilization membranes. Biochem. Mol. Biol. Int. 1995, 36 (1), 59–66. (9) Romero-Calvo, I.; Ocon, B.; Martinez-Moya, P.; Suarez, M. D.; Zarzuelo, A.; Martinez-Augustin, O.; de Medina, F. S. Reversible Ponceau staining as a loading control alternative to actin in Western blots. Anal. Biochem. 2010, 401 (2), 318–20. (10) D’Souza, A.; Scofield, R. H. Protein stains to detect antigen on membranes. Methods Mol. Biol. 2009, 536, 433–40. (11) Gallagher, S.; Winston, S. E.; Fuller, S. A.; Hurrell, J. G. Immunoblotting and Immunodetection. Current Protocols in Immunology; 2008; 83, 8.10.1-8.10.28. 1419
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