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Conditions that allow for effective transfer of membrane proteins onto nitrocellulose membrane in Western blots Priyanka D. Abeyrathne and Joseph S. Lam
Abstract: A major hurdle in characterizing bacterial membrane proteins by Western blotting is the ineffectiveness of transferring these proteins from sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) gel onto nitrocellulose membrane, using standard Western blot buffers and electrophoretic conditions. In this study, we compared a number of modified Western blotting buffers and arrived at a composition designated as the SDS–PAGE-Urea Lysis buffer. The use of this buffer and specific conditions allowed the reproducible transfer of highly hydrophobic bacterial membrane proteins with 2–12 transmembrane-spanning segments as well as soluble proteins onto nitrocellulose membranes. This method should be broadly applicable for immunochemical studies of other membrane proteins. Key words: bacterial membrane, membrane protein, Western blotting, Pseudomonas aeruginosa, lipopolysaccharide, Wzydependent pathway, WaaL. Re´sume´ : Un obstacle majeur a` la caracte´risation des prote´ines membranaires bacte´riennes par buvardage Western est l’inefficacite´ de leur transfert du gel SDS–PAGE a` la membrane de nitrocellulose dans des tampons de buvardage Western et des conditions d’e´lectrophore`se standards. Dans cette e´tude, nous avons compare´ un certain nombre de tampons de buvardage Western modifie´s et avons de´termine´ la composition d’un tampon de lyse SDS–PAGE-ure´e. L’utilisation de ce tampon sous des conditions spe´cifiques a permis de transfe´rer de fac¸on reproductible des prote´ines membranaires bacte´riennes hautement hydrophobes posse´dant de 2 a` 12 traverse´es membranaires, ainsi que des prote´ines solubles, sur des membranes de nitrocellulose. Cette me´thode devrait eˆtre largement applicable lors d’e´tudes immunochimiques d’autres prote´ines membranaires. Mots-cle´s : membrane bacte´rienne, prote´ine membranaire, buvardage Western, Pseudomonas aeruginosa, lipopolysaccharide, patron de´pendant de Wzy, WaaL. [Traduit par la Re´daction]
Characterization of proteins often relies on an effective method for detecting and identifying a protein of interest. One of the most frequently used methods for this type of study is the electrophoretic transfer of proteins that have been resolved by a sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) slab gel onto a nitrocellulose (NC) membrane. Identification of a specific protein band can be achieved by either radiolabeling or immunochemical probing (Towbin et al. 1979). This method is more commonly known as ‘‘Western blotting’’ (Burnette 1981). Since this technique was published, many modifications have been described (Gershoni and Palade 1983; TowReceived 13 November 2006. Revision received 3 January 2007. Accepted 9 January 2007. Published on the NRC Research Press Web site at cjm.nrc.ca on 25 May 2007. P.D. Abeyrathne and J.S. Lam.1 Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON N1G 2W1, Canada. 1Corresponding
author (e-mail:
[email protected]).
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bin et al. 1984; Dunn 1986), but none of the modifications was designed specifically for working with membrane proteins. The proteins in a SDS–PAGE gel are normally denatured, but their overall structure after transfer to NC is not known. The state of protein folding is particularly important when a monoclonal antibody, which recognizes only a single epitope, is used as the immunoprobe. Partial renaturation might occur during transfer from a SDS–PAGE gel to NC if no denaturant is present in the transfer buffer. Inherent in each Western blotting step are factors that may affect the overall efficiency of the transfer technique. Furthermore, success in the electroelution of proteins depends on a number of factors, including the pore sizes in the matrices of the polyacrylamide gel, the molecular weight and net charge of the polypeptides, and the electrical current applied. In the electrotransfer step, immobilization of proteins onto NC membranes is mainly due to hydrophobic interactions (Lin and Kasamatsu 1983). Thus, the presence of reagents that modify these interactions, such as nonionic detergents, could affect the immobilization processes. Apolar solvents have been used for the electrotransfer pro-
doi:10.1139/W07-007
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Table 1. Buffers used in the Western blotting experiments. Buffer Buffer Buffer Buffer Buffer Buffer
name I (Tris–glycine blotting buffer) II III (carbonate buffer) IV V (SP-UL buffer)
Buffer compositiona,b 25 mmol/L Tris, 192 mmol/L glycine, pH 8.3, in 20% methanol (v/v) 25 mmol/L Tris, 192 mmol/L glycine, 0.01%–2% SDS (m/v), pH 8.3, in 20% methanol 10 mmol/L NaHCO3, 3 mmol/L Na2CO3, pH 9.9, in 20% methanol 25 mmol/L Tris, 192 mmol/L glycine, pH 8.3 1:1 mix of 1 SDS running buffer (12 mmol/L Tris, 96 mmol/L glycine, 0.1% SDS) and urea lysis buffer (10 mmol/L Na2HPO4, 1% b-mercaptoethanol (v/v), 1% SDS, 6 mol/L urea)
a The same electrophoretic blotting transfer condition was used for each of the buffers listed. The current used was 180 mA for 45 min, and the temperature was maintained below 35 8C by placing an ice pack inside the electrophoresis chamber. b Note that methanol was added as a percentage of the total volume (v/v), SDS was added at a particular percentage (m/v), and b-mercaptoethanol was added according to the percentage shown (v/v).
cesses presumably to favor the dissociation of SDS and to expose the hydrophobic groups of the polypeptides (Lin and Kasamatsu 1983). Our laboratory is interested in investigating the functions of a group of membrane proteins that are essential for the biosynthesis of lipopolysaccharide (LPS) of Pseudomonas aeruginosa. Several proteins in this organism, including WaaL, Wzy, Wzz1, Wzz2, and Wzx, have been proposed to form a membrane complex (Rocchetta et al. 1999) and participate in the Wzy-dependent LPS-assembly pathway (Whitfield 1995). By using the Kyte and Doolittle hydropathy plot (Kyte and Doolittle 1982) and the transmembrane hidden Markov model (TMHMM Server v. 2.0) online servers, we found that all of these proteins from P. aeruginosa are membrane proteins possessing multiple transmembranespanning segments (TMS). WaaL, an O-antigen ligase encoded by pa4999 in the P. aeruginosa PAO1 genome (Stover et al. 2000; Abeyrathne et al. 2005), possesses 11 potential TMS. Wzy, an O-antigen polymerase encoded by pa3154 (de Kievit et al. 1995), and Wzx, an O-antigen flippase encoded by pa3153 (Burrows and Lam 1999), each possess 12 potential TMS. Wzz1 and Wzz2, O-antigen chain-length regulators encoded by pa3160 and pa0938, respectively (Daniels et al. 2002), possesses two potential TMS. Despite the fact that membrane proteins are notoriously difficult to express, we were able to successfully over-express all five of the aforementioned membrane proteins. In this study, we describe an approach that allows reproducible transfer of these membrane proteins for immunochemical characterization in Western blotting. The following conditions were used throughout this study and WaaL was used as a reference protein. The amount of protein used per sample was quantified based on comparison with bovine serum albumin (BSA) standards in the range of 0.5–5 mg run on SDS–PAGE gels and recorded by gel densitometry using a Gel DocTM instrument (Bio-Rad, Hercules, California). For each membrane protein, 3–4 mg of crude protein extract from bacterial cells expressing the earliernoted membrane proteins was used. The discontinuous slab gel and buffer system described by Laemmli (1970) was used, and gels were cast with a 0.75 mm thick space bar and 12% polyacrylamide using a Bio-Rad mini-gel system. Protein samples were prepared for SDS–PAGE by heating at either 37 8C for 30 min or at 100 8C for 10 min in a SDS sample buffer containing 63 mmol/L Tris–HCl, pH 6.8, 3% SDS, 5% b-mercaptoethanol, 0.01 mg/mL bromophenol blue, and 10% glycerol. Electrophoresis was per-
formed at 100 V, and protein samples were run until the bromophenol blue tracking dye reached the bottom of the separating gel. In each experiment, protein samples were run on duplicate gels. One of the gels was stained with Coomassie BlueTM R-250 (Sigma-Aldrich, St. Louis, Missouri) or SimplyblueTM safestain (Invitrogen, Carlsbad, California), while the other was used for blotting and was subsequently stained to assess the efficiency of the electrotransfer procedure. Electrophoretic transfer of proteins onto NC membranes was performed in a Bio-Rad transblot cell. The current used was 180 mA for 45 min and the temperature was maintained below 35 8C by placing an ice pack inside the electrophoresis chamber. After blotting, the NC membranes were stained with Ponceau S (Sigma-Aldrich) to visualize the transfer of proteins from the gels to the membranes. The following steps were performed at room temperature. The membranes were rinsed with deionized water (dH2O) followed by washing twice for 10 min each with Tris-buffered saline (TBS, composed of 10 mmol/L Tris–HCl (pH 7.5) and 150 mmol/L NaCl). The membranes were then blocked by treating for either 1 h or overnight with 3% (m/v) BSA in TBS. After that, the membranes were washed once for 10 min in TBS–Tween–Triton buffer (TBS-TT) containing 20 mmol/L Tris–HCl (pH 7.5), 500 mmol/L NaCl, 0.05% (v/v) Tween 20 (Sigma-Aldrich), 0.2% (v/v) Triton X-100 (Sigma-Alrich), followed by washing twice for 10 min each time in TBS buffer. The NC membranes were then incubated overnight with an anti-His5 monoclonal antibody (Qiagen, La Jolla, California) (used at 1/2000 dilution in TBS with 3% BSA). The membranes were rinsed for 10 min in TBS-TT at room temperature followed by two 10 min rinses in TBS buffer. Next, the blots were incubated overnight (16 h) with goat anti-mouse F(ab’)2-alkaline phosphatase conjugated secondary antibody (Jackson Immuno Research, West Grove, Pennsylvania) at a 1/2000 dilution in TBS with 3% BSA. The blots were washed (10 min per wash) once with TBS-TT buffer and twice with TBS buffer. The membranes were then developed using a substrate containing 0.333 mg/mL nitroblue tetrazolium (NBT; Sigma-Aldrich) and 0.15 mg/mL 5-bromo-4chloro-3-indolyl phosphate (Sigma-Aldrich) in 0.1 mol/L bicarbonate buffer (pH 9.8). The colorimetric reaction was stopped by rinsing the membranes thoroughly with ultrapure water obtained from a Super-QTM water purification system (Millipore, Billerica, Massachusetts). Five different Western blot buffer systems (Buffers I–V) were examined in the electroblotting step and their composi#
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Fig. 1. Blotting of WaaL onto nitrocellulose (NC) membrane using various buffers. Panel A shows a sodium dodecyl sulfate – polyacrylamide gel electrophoresis gel of proteins stained with SimplyblueTM safestain before blotting. Panel B shows a stained gel after blotting. Panel C shows a Western blot reacted with anti-His5 antibody and developed with alkaline phosphatase-conjugated secondary antibody and substrate. Section I — use of Buffer I (standard Tris–glycine transfer buffer) as a Western blot. Section II — examination of the effectiveness of Buffer III (carbonate transfer buffer). Section III — effect of Western blotting transfer using Buffer V (SP-UL buffer). Panels A–C from sections I–III: lane 1 contains total membrane protein fraction with empty vector pVLT31 and lane 2 contains total membrane protein fraction of pVLT31-WaaL. Arrows in panels A and B indicate expressed WaaL. Arrow in panel C, section III, indicates the transferred WaaL. M, prestained molecular weight standards, which are marked on the left-hand side of the figures in each section.
tions are described in Table 1. Buffer I, designated as ‘‘Tris– glycine blotting buffer,’’ is the standard blotting buffer used for Western immunoblotting. Initial attempts to transfer the WaaL protein onto a NC membrane using the standard Buffer I and Western blotting protocol did not meet with success. No effective transfer was observed and WaaL was found to remain in the SDS–PAGE gel (Fig. 1, section I, panel B). The blotting efficiency was unchanged when other conditions were used; which included extending the blotting time from the standard 45 min to 2 h, 4 h, and overnight,
and a range of temperatures, from 4 8C to room temperature. Thus, while the standard Western blot method is effective for transferring most proteins from SDS–PAGE gels onto a membrane support, our results are consistent with the findings in a recent report by Drew et al. (2006), who showed that proteins containing a high proportion of hydrophobic amino acids as well as TMS are not easily transferred to a NC membrane during the blotting step of the Western blot technique. Another cause for failure in transferring membrane proteins to NC membranes may be that no SDS was #
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Fig. 2. Effect of Buffer V on protein blotting efficiency and immunochemical detection of four different membrane proteins. Panel A shows a sodium dodecyl sulfate – polyacrylamide gel electrophoresis gel of proteins stained with SimplyblueTM safestain before blotting. Panel B shows a stained gel after blotting. Panel C shows a Western blot reacted with anti-His5 antibody and developed with substrate. Section I — Wzz1 and Wzz2; lane 1 in panels A–C contains the total membrane protein fraction of Escherichia coli with empty vector pET28a; lanes 2 and 3 contain the total membrane fractions of E. coli with pET28a-Wzz1 and pET28a-Wzz2, respectively. Section II — Wzy; lane 1 contains the total membrane protein fraction of Pseudomonas aeruginosa with empty vector pVLT31; lane 2 contains total membrane protein fraction of P. aeruginosa with pVLT31-Wzy. Section III — Wzx; lane 1 contains the total membrane protein fraction of E. coli with empty vector pQE80; lane 2 contains the total membrane protein fraction of E. coli with pQE80-Wzx. Arrows in panel C, sections I–III, are transfered Wzz2, Wzz1, Wzy, and Wzx proteins, respectively. M, prestained molecular weight standards.
used in the standard Western blot transfer buffer (Nielsen et al. 1982; Dunn et al. 1985). In light of this, various concentrations of SDS were added to the transfer buffer and the modified buffers were used in our experiments. By switching to Buffer II (Table 1), which has the same composition as Buffer I differing only on the varying amounts of SDS (0.01%–2%) being added, we could not observe any visible improvement on the blotting of WaaL from the gel onto the NC membrane. Buffer III is a carbonate buffer (Table 1), which has an alkaline pH at 9.9 and has been used to allow more efficient Western transfer of the g subunits of F1-ATPase of Escherichia coli, which has an isoelectric point of about 8.9 (Dunn 1986). The isoelectric point of WaaL (pI = 9.95) is also
close to 10, so we also tested Buffer III in electrotransfer of protein onto NC membrane. The amount of protein left in the SDS–PAGE gel after the electroblotting step, however, was similar to when Buffer I was used (Fig. 1, section II, panel B). After development with appropriate antibodies and substrate, no protein band with the apparent mass of WaaL could be discerned in the NC blot (Fig. 1, section II, panel C). It is not uncommon that proteins of larger molecular masses may not be completely eluted out of the gel during the Western transfer step. However, this was likely not the case here since WaaL has a relatively low molecular mass of 44 398.68 Da. To alleviate problems in protein transfer that might have been caused by the presence of methanol, nylon membranes were used in#
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Fig. 3. Use of Buffer V for transfer of soluble proteins. Panel A shows a sodium dodecyl sulfate – polyacrylamide gel electrophoresis gel of proteins stained with SimplyblueTM safestain before blotting. Panel B shows a stained gel after blotting. Panel C shows a Western blot reacted with anti-His5 antibody and developed with alkaline phosphatase-conjugated secondary antibody and substrate. Section I — WbpD; lane 1 in panels A–C contains the soluble protein fraction of Escherichia coli with empty vector pET28a; lane 2 contains the soluble fraction of E. coli with pET28a-WbpD. Section II — WbjB; lane 1 contains the soluble protein fraction of E. coli with empty vector pET28a; lane 2 contains the soluble protein fraction of E. coli with pET28a-WbjB. Arrows in panel C, sections I and II, point to blotted WbpD and WbjB proteins, respectively. M, prestained molecular weight standards.
stead of NC membranes. The use of nylon membranes usually does not require methanol in a Western transfer buffer. This buffer was designated as Buffer IV (Table 1), and the composition is identical to that of Buffer I except that it lacked methanol. However, no improvement was observed in transferring of WaaL onto the nylon membrane using Buffer IV (data not shown). Further, we tested the use of polyvinylidene fluoride membrane in the blotting step but did not observe any improvement over NC or nylon membranes. To determine whether pretreatment of proteins in SDS gels before electroblotting could improve the transfer of WaaL from the gel to the NC membrane, we immersed the SDS–PAGE gel in 50 mmol/L Tris–HCl, pH 7.4, for 1 h at room temperature, followed by Western transfer using the carbonate blotting buffer (Buffer III). No improvement in the transfer of WaaL onto the NC membrane was observed. Blank and co-workers (1982) described the conditions for renaturing ribonuclease in gels by using a renaturation buffer containing 25% isopropanol. This approach was followed but no improvement in the transfer of WaaL onto NC membranes could be observed. Bowen and coworkers (1980)
suggested that the use of 4 mol/L urea could aid the renaturation of proteins in gels. We presoaked a SDS–PAGE gel containing resolved membrane proteins including WaaL in a buffer containing 4 mol/L urea in 50 mmol/L Tris–HCl, pH 7.4, for 1 h, followed by 30 min in a 50 mmol/L Tris– HCl buffer before electrotransfer (in Buffer I) of the proteins to the NC membrane. Despite this treatment, no improvement in the transfer of WaaL onto the NC membrane could be observed. Another condition that was carried out was a renaturation procedure using 20% glycerol in Tris–glycine buffer without SDS. Again, no improvement in the transfer of WaaL onto the NC membrane could be observed (data not shown). Since WaaL could migrate in SDS–PAGE gels with a relatively consistent mobility as compared with the protein standards being used, it became apparent that the running buffer used in SDS–PAGE was effective in resolving WaaL from other molecules found in the crude bacterial membrane protein extracts. Therefore, the standard SDS–PAGE running buffer was used in the Western blot transfer procedure, but we did not observe a significant improvement in the #
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transfer of WaaL onto NC membrane as compared with the various conditions and buffers described above (Table 1). Next, SDS–PAGE running buffer was modified by mixing it at a ratio of 1:1 (v/v) with urea lysis buffer (Amer and Valvano 2000), which is normally used to lyse cells for isolating proteins from bacterial cells. This buffer was designated as Buffer V (also called SP-UL buffer, i.e., SDS– PAGE-Urea Lysis buffer — Table 1) and the use of this buffer gave a striking improvement in the electrotransfer of WaaL from SDS–PAGE gels onto NC membranes. Complete transfer of WaaL from the gel to the NC membrane was achieved since no protein band at 44 398.68 Da (the mass of WaaL) could be detected in the SDS–PAGE gel following Western transfer (Fig. 1, section III, panel B). The immunoblot showed a single band at an apparent molecular mass of 44 kDa (Fig. 1, section III, panel C). The efficiency of SP-UL buffer to mediate blotting transfer of WaaL onto NC was highly reproducible. To determine the general applicability of the new SP-UL buffer for Western blotting, transfer of other membrane proteins (Wzy, Wzx, Wzz1, and Wzz2) onto NC membranes was attempted. Each of these proteins has been successfully overexpressed and subjected to electrophoresis by SDS– PAGE. After staining with SimplyblueTM safestain each of these four proteins was clearly visible (Fig. 2, panel A). Using Buffer V (SP-UL buffer) in the blotting step, all of the membrane proteins were effectively transferred to the NC membrane. Staining of the post-Western transferred SDS– PAGE gel with SimplyblueTM safestain showed that it lacked the respective protein bands (Fig. 2, panel B). Two different soluble proteins, WbpD and WbjB, from P. aeruginosa were used as controls to determine the efficiency of Buffer V (SP-UL buffer) in transferring soluble proteins. WbpD and WbjB have been described previously by our group (Kneidinger et al. 2003; Wenzel et al. 2005) and these have proteins been shown to be easily transferred to NC membrane using Buffer I and standard Western blotting conditions. As anticipated, after Western transfer of both soluble proteins onto NC membranes, protein bands at apparent molecular mass of 23 kDa (WbpD) and 40.7 kDa (WbjB) that were reactive with anti-His5 monoclonal antibody could be visualized on the immunoblot (Fig. 3). Prior to this study, there was no established procedure for the effective transfer of integral membrane proteins from SDS–PAGE gels onto NC membranes during Western blotting. Although a variety of conditions were used to modify both the blotting procedures and the content of the standard Western blot buffer, none of the conditions could yield effective transfer of the highly hydrophobic membrane protein WaaL, which has 11 TMS, onto NC membrane. In contrast, the use of the new SP-UL buffer (Buffer V) allows complete transfer of the WaaL protein (Fig. 1, section III, panel B) as well as the other membrane proteins, Wzy (12 TMS), Wzx (12 TMS), Wzz1 (2 TMS), and Wzz2 (2 TMS), from SDS– PAGE gels onto NC membranes (Fig. 2, panel B). In conclusion, we have clearly demonstrated that the use of this novel Western SP-UL buffer (Buffer V) is highly efficient in the blotting of the membrane proteins onto NC membranes, and the process has proven to be reliable and reproducible. The improvements appear to result from better transfer of membrane proteins as well as soluble proteins
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and corresponding proteins are easily detectable in the immunoblots after reacting these with an appropriate antibody probe, enzyme-conjugated secondary antibody, and substrate. The SP-UL blotting buffer (Buffer V) should be broadly applicable when other membrane proteins are being examined using Western blotting, and should contribute to a better understanding of the structure and function of membrane proteins.
Acknowledgements This work was supported by an operating grant to J.S.L. from the Canadian Cystic Fibrosis Foundation (CCFF) and by a Research Tools and Instruments grant (263786-03) from the Natural Sciences and Engineering Research Council of Canada for the purchase of a Millipore Super-QTM water purification system. P.D.A. is a recipient of a CCFF fellowship and J.S.L. holds a Canada Research Chair in Cystic Fibrosis and Microbial Glycobiology. The authors also thank Craig Daniels for his critical reading of the manuscript.
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