Created: 1st September 1999, last updated: 12th November 1999, © 1999 ABRF
Daniel R. TerBusha and Peter Novickb
aDepartment of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, MD; and bYale University School of Medicine, Department of Cell Biology, New Haven, CT
Reprinted from the electronic version of JBT available at http://www.abrf.org/JBT/JBT.html accession #0018.
A high concentration of substrate protein facilitates efficient in-gel protease digestion of samples prior to high-performance liquid chromatography (HPLC) separation and peptide microsequencing. To facilitate quantitative recovery and concentration of proteins separated on large sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, a method of concentrating proteins using funnel tube (FT) gels was developed. This method allows the use of very large sample sizes (up to 5 mL of sample buffer and gel slices per FT gel) and yielded quantitative recovery when 5 µg of a protein standard was concentrated. FT gel protein concentration was developed during the purification of several components of the yeast Exocyst complex. The yeast Exocyst complex was immunoprecipitated, and its components were separated by sequential rounds of SDS-PAGE followed by FT gel protein concentration. The individual protein components were concentrated a total of 676-fold into 71-mm3 gel slices. The resulting in-gel protein concentrations were at least 0.2 µg/mm3. The FT gel protein concentration methodology should be useful whenever a small amount of protein must be concentrated from a very large gel or sample volume prior to protease digestion and HPLC or other characterization techniques. (J Biomol Tech 1999;10:149-152)
Key words: protein concentration, funnel tube gels, Saccharomyces cerevisiae, SEC genes, Exocyst, secretory pathway.
Address correspondence and reprint requests to Daniel R. TerBush, Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814 (email: danter1@aol.com).
A significant problem in proteolytic digestion of large sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-purified proteins is obtaining a high in-gel protein concentration. A high in-gel protein concentration greatly facilitates complete proteolytic digestion.1 The slab funnel gel method uses SDS-PAGE to concentrate protein in a gel slice, thereby avoiding the need to use other protein-concentrating methods.2 However, this technique is limited in capacity, and the gels have a tendency to leak the applied sample from the gel sandwich due to inefficient acrylamide polymerization at the gel-spacer interface. The funnel tube (FT) gel method for concentrating proteins (described later) does not leak and efficiently concentrates protein from very large sample volumes (up to 5 mL of gel slices and buffer) into a small gel volume containing high in-gel protein concentration.
This method was developed during the purification of a multiprotein complex, the Exocyst, which is required for exocytosis in Saccharomyces cerevisiae.3,4 Before this study, the Exocyst was known to contain at least eight polypeptides, including Sec6p, Sec8p and Sec15p.4 The Exocyst complex proteins were isolated by immunoprecipitation and separated by SDS-PAGE. The unknown Exocyst components were then concentrated by FT gel protein concentration. The high recovery of protein allowed for a second round of separation and concentration which completely removed contaminating IgG from the proteins of interest. Sufficient quantities of the unknown Exocyst proteins were recovered for efficient in-gel proteolytic digests, HPLC, mass spectrometry, and peptide microsequencing.
Each FT was constructed by a scientific glass-blower from a 10-mm (outside diameter) heavy-walled glass tube (inside diameter ~5.5 mm) to which a 25-mm thin-walled glass tube was affixed. The top 25-mm tube was cut to about 2 cm high to form a large bowl-shaped funnel above the 10-mm tube. The bottom 10-mm tube was then cut to about 19 cm long. In addition, a Maria (ie, a bulge) was placed in the neck of the tube 4.5 cm from the top so that the FT would rest on this bulge when placed in the electrophoresis tank (Fig. 1).
FIGURE 1. Schematic diagram of a funnel tube (FT) gel. Sample buffer or a mixture of gel slices and sample buffer are loaded into the top of the FT gel. Proteins concentrate at the stacking gel-running gel interface and enter the running gel just behind the lagging edge of the sample buffer dye.
The electrophoresis tank used in this study was improvised from a 25-year-old Bio-Rad tube gel apparatus (Hercules, CA, http://www.bio-rad.com/index.html) capable of handling 11-mm-outside-diameter tubes, but most university shops should be able to fashion a workable unit. Additionally, a Bio-Rad Model 175 tube cell could be used, but with smaller capacity tubes (8 mm outside diameter).
The constructed FTs were used to concentrate protein from sample buffer-gel slice mixtures. First, 5-cm-tall running and stacking gels were sequentially poured into the FTs and allowed to polymerize (1.4 mL of unpolymerized gel mixture each). After the gels polymerized, the FT was glued into the electrophoresis tank using RTV Multi-Purpose Sealant Model 732 (Dow Corning, Midland, MI). Up to 5 mL of protein contained in sample buffer or a sample buffer-gel slice mixture is then loaded into the top of the FT gel. For a FT gel with the dimensions described previously, the voltage during the electrophoresis run was limited to 150 V and the current was limited to 7 mA per tube to avoid heating. The concentrated protein elutes from the gel slices and stacks at the interface of the stacking and running gels. The protein concentrates at the stacking gel-running gel interface and enters the running gel just behind the dye front. When the trailing edge of dye was about 4 or 5 mm into the top of the running gel, the electrophoresis was stopped. Following an electrophoresis run, each FT was merely pulled hard to remove it from the tank, although sometimes the sealant had to be partially cut away. The running gel can then be easily extruded from the FT by placing the bottom 10 cm of the tube under hot running water until the glass is warm to the touch. The gel can then be pushed out of the bottom of the FT with a 5-mm-diameter glass rod. The top 4 or 5 mm of the running gel, which contains the concentrated protein, was then excised.
FT gels were used to concentrate 5- and 15-µg samples of beta-galactosidase, and the efficiency of protein recovery was quantitated. The protein samples were first boiled in 6 mL of sample buffer (3% SDS, 10% glycerol, 2% 2-mercaptoethanol, 2% bromophenol blue, 0.125 M Tris-HCl, pH 6.8) and applied separately to single-well 16-cm X 14-cm X 1.5-mm, 7% acrylamide SDS-PAGE slab gels. After "separation," each gel was stained with Coomassie blue for 10 minutes (0.125% Coomassie blue, 50% MeOH, 10% acetic acid) and destained (one wash with 50% MeOH + 10% acetic acid followed by several washes with 5% MeOH + 7% acetic acid). The beta-galactosidase protein band was excised. The gel slices were then incubated at room temperature with several changes of water until the pH was between 5 and 6. They were then rocked with an equal volume of 2X sample buffer for at least 2 hours and frozen overnight. The sample buffer-gel slice mixture was thawed and placed on a rocking platform for another 2 hours before loading the FT gel. After completion of the FT gel protein concentration, the resulting gel slice was fixed with three changes of solution containing 50% MeOH and 10% acetic acid. Protein recovery was quantitated by amino acid composition analysis performed by the W. M. Keck Laboratory, Yale University.
The Exocyst complex proteins were purified by immunoprecipitation and sequential rounds of slab gel-FT gel SDS-PAGE electrophoresis. The Exocyst complex proteins were isolated from a lysate containing 6.4 g total protein by immunoprecipitation with 3.6 mL of ascites (anti-c-myc, monoclonal antibody 9E10) as previously described.3,4 The immunoprecipitate was boiled in 48 mL of sample buffer to liberate the immunoisolated proteins. One fourth of the total immunoextract (12 mL) was multiloaded into a 1-well 16-cm X 14-cm X 1.5-mm, 7% acrylamide SDS-PAGE slab gel. Four gels were used for the initial electrophoresis. The slab gels were stained and destained as described previously. Eight Exocyst complex polypeptides specifically co-immunoprecipitate (Fig. 2), as previously shown elsewhere.3,4
FIGURE 2. Characteristic protein pattern of an Exocyst complex immunoprecipitate. Shown are the Coomassie blue-stained polypeptides that co-immunoprecipitate with c-myc-tagged Sec8p with anti-c-myc antibody. This is an image of one of the four Coomassie blue-stained gels from the first SDS-PAGE protein separation. The gel is heavily overloaded with protein--in particular, IgG. Bands B (c-myc-Sec8p), C (Sec15p), and G (Sec6p) have been previously identified.4 Bands A, D, E, F, and H were previously shown to co-immunoprecipitate with c-myc-Sec8p, but their identities were unknown prior to their purification and peptide sequencing.3
The protein bands corresponding to positions A, D, E, F, and H were excised from the gels, neutralized with water, and equilibrated with an equal volume of fresh 2X sample buffer. The proteins contained in each band were then concentrated with FT gels as described earlier.
The first round of SDS-PAGE slab gel separation failed to cleanly separate the eight Exocyst complex polypeptides from each other because of the extreme overloading of the initial slab gel. The proteins concentrated from the first slab gel (bands A, D, E, F, and H) actually contain a mixture of Exocyst components and IgG (Fig. 3). A second round of SDS-PAGE slab gel separation was necessary to resolve the individual components. The gel plugs from the first FT concentration gel were chopped into 1-mm cubes and equilibrated with an equal volume of fresh 2X sample buffer for 2 hours. The gel cubes and sample buffer were then loaded into a second 16-cm X 14-cm X 1.5-mm, 7% acrylamide SDS-PAGE slab gel (10-well comb) and electrophoresed. The second separating gel was stained and destained as described earlier. The second separating gel gave a clean separation of all the concentrated proteins. One lane each of the second separating gel for bands D and E is shown in Figure 3.
FIGURE 3. Second SDS-PAGE gel separation. The bands corresponding to positions D and E from the first slab gel (see Fig. 2) were concentrated with FT gels and reseparated on a second slab gel. Shown is the Coomassie blue-stained second separating gel. For bands D and E, the material concentrated from the first slab gel was contaminated with other Exocyst complex proteins and IgG. The first separating gel was heavily overloaded, resulting in proteins from a given band smearing and contaminating adjacent protein bands. The second separating gel cleanly resolves the mixture of proteins.
Gel slices from the second separating gel corresponding to a given unknown were then processed exactly as described earlier and concentrated in a final FT gel. The resulting 5.5-mm X 3-mm gel slices were fixed overnight with at least three changes of solution containing 50% MeOH and 10% acetic acid. The gel slices containing the polypeptides of bands A, D, E, F, and H were then given to the W. M. Keck Laboratory for amino acid analysis, trypsin and/or lys-C in-gel digestion, HPLC separation, mass spectrometry, and microsequencing of the proteolytic peptides.
We used FT gels to concentrate known amounts of beta-galactosidase from SDS-PAGE slab gel slices to test the efficiency of the FT gel protein concentration method. Gel slices from the SDS-PAGE slab gels containing either 5 or 15 µg of beta-galactosidase were concentrated with FT gels into 71-mm3 gel plugs. We recovered an average of 100% (n = 3) of the 5-µg of protein standard and 65% (n = 2) of the 15 µg of protein standard. Recovery was less efficient with larger (15 µg) than with smaller (5 µg) protein samples, possibly as a result of overloading of the tube gel resulting in poor stacking. For the 5 µg and 15 µg protein standards, this yielded protein concentrations of 0.07 µg/mm3 and 0.14 µg/mm3 of gel volume, respectively. For both, the protein was concentrated about 70-fold (from 5000 mm3 to 71 mm3).
We next applied the technique of sequential SDS-PAGE separating gel-FT concentrating gel electrophoresis to the purification of the uncharacterized components of the yeast Exocyst complex (Figs. 2 and 3). We estimate that 640 µg of protein corresponding to bands D and E are present in the 6.4 g of starting lysate used for their purification (each Exocyst protein is about 0.01% of total cellular protein).3 In the large-scale immunoprecipitation used for these purifications, approximately 5% of the total c-myc-Sec8p was immunoprecipitated (data not shown).3 Because the Exocyst complex proteins are present in a 1:1 weight ratio to Sec8p,3 the sample buffer loaded onto the first separating gels contained about 32 µg of the band D and E proteins. We recovered 20 µg of band D protein (62%) and 9.7 µg of band E protein (30%) after two SDS-PAGE separating gel and FT concentrating gel electrophoresis cycles. The yield of purified protein from the starting material (640 µg) was 3.1% for band D and 1.5% for band E, with most of the protein being lost by the inefficient immunoprecipitation. The concentration of protein in the final gel slices was 0.42 and 0.20 µg/mm3, respectively. The results for the other Exocyst components were comparable to those obtained for bands D and E (data not shown).
We were able to obtain peptide sequence from bands D, E, F, and H and determine the identity of band A by mass pattern matching.3 Comparison of the peptide sequences to a translation of the Saccharomyces cerevisiae genome database, in conjunction with our own cloning and sequencing results, identified D as Sec5p, E as Sec10p, F as a C-terminal breakdown product of Sec3p, and H as a novel protein of 70 kd which we called Exo70p.3 Band A was identified as full-length Sec3p by mass pattern matching.3
We have described a method of concentrating protein using FT gels and applied this method to the purification of the unknown protein components of a multiprotein complex required for exocytosis in yeast. This method has several advantages: very large starting sample volume; absence of leakage from FT gels; high resulting in-gel protein concentrations; and quantitative protein recovery from dilute samples. The quantitative recovery of the 5-µg beta-galactosidase protein standard suggests that this method could be applied with equal success to samples containing much lower protein amounts.
The high efficiency of protein recovery allowed the use of multiple cycles of SDS-PAGE protein separation and FT gel protein concentration. This combination was a powerful aid in obtaining highly concentrated, pure Exocyst component proteins free of contaminating IgG. We were able to concentrate the purified Exocyst proteins a total of 676-fold with the FT gel method. The minimum in-gel protein concentration obtained (0.20 µg/mm3) was well above the desired minimum for efficient proteolytic digestion (0.03 µg/mm3).1 The concentrated protein was suitable for in-gel protease digestion, HPLC separation of the resulting peptides, and peptide microsequencing.
We would like to thank Ralph Stevens and Alan Brown for making the FTs. We would also like to thank all the members of the W. M. Keck Sequencing Facility at Yale for their help and patience, in particular Kathryn Stone, Myron Crawford, Edward Papacoda, Mary Lo Presti, and Kenneth Williams. Finally, we would like to thank James Jamieson for the use and gift of the Bio-Rad tube gel tank. This research was supported by National Institutes of Health grant GM35370, by Uniformed Services University of the Health Sciences grant RO71EK, and by a grant from the Edward Mallinckrodt, Jr. Foundation. The opinions and assertions contained herein are private ones of the authors and are not to be construed as official or reflecting the views of the Department of Defense or the Uniformed Services University of the Health Sciences.
1. Williams KR, Stone KL. In gel digestion of SDS PAGE-separated proteins: observations from internal sequencing of 25 proteins. In Crabb JW (ed): Techniques in Protein Chemistry, vol 6. San Diego: Academic Press, 1995:143-152.
2. Lombard-Platet G, Jalinot P. Funnel-well SDS-PAGE: a rapid technique for obtaining sufficient quantities of low-abundance proteins for internal sequence analysis. Biotechniques 1993;15:669-672.
3. TerBush DR, Maurice TM, Roth D, Novick P. The Exocyst is a multiprotein complex required for exocytosis in Saccharomyces cerevisiae. EMBO J 1996;15:6483-6494.
4. TerBush DR, Novick P. Sec6, Sec8, and Sec15 are components of a multisubunit complex which localizes to small bud tips in Saccharomyces cerevisiae. J Cell Biol 1995;130:299-312.