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Protein Recovery from Denaturing or Native Gels

D. Sheer, Ph.D., Millipore Corporation
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is the most widely used analytical technique for protein separation. The ability to obtain structural information and characterize proteins, using picomole amounts of sample, has also increased the utility of SDS-PAGE as a micropreparative purification technique. The major advantage of SDS-PAGE is high resolution. However, sample recovery and preparation can compromise overall results. Blotting onto support such as polyvinylidene difluoride (PVDF) has become one of the primary methods of choice in total purification of proteins for structural information. However, in cases of microsequence analysis, in vivo NH2-terminal blocking requires that the electroblotted protein be cleaved into fragments which must then be individually purified before sequencing. The low protein recoveries when using these techniques and the inability to work with biologically active enzymes in solution limits the usefulness of electroblotting to obtain structural information or to characterize gel-purified proteins.

Various elution methods and devices for protein recovery have been developed. These include:

  1. Electroelution into membrane traps from gel slices.

  2. Continuous electroelution during the running of gels.

  3. Diffusion out of homogenized gel slices.
However, important drawbacks limit the usefulness of these methods, e.g. large recovery volumes, contaminating salts and detergents as well as time-consuming labor-intensive procedures. By contrast, sample processing with Amicon's combined Microcon®/MicropureTM device facilitates extraction, removal of buffer and detergents. It eliminates multiple handling of protein solutions. Furthermore, gel homogenization followed by high centrifugal forces (14,000 × g), improves extraction. Micropure and Microcon allow rapid sample recovery with removal of salts and detergents by exchange washes before retrieving the concentrated protein. This report presents protocols with examples that demonstrate the use of this product combination in preparing purified protein from gels for subsequent study.


Materials and Methods

Native Page
Non-denaturing, non-reducing PAGE is performed with 0.5 mm thick 10% resolving gels, using the Tris-Glycine buffer system. ß-galactosidase (grade VI from E. Coli), glucose-6-phosphate dehydrogenase and alkaline phosphatase (Sigma Chemical Company) is prepared as a stock solution in 20 mM Tris/HCl, pH 7.3. Amounts ranging from 1 to 15 µg of each enzyme are diluted in equal volumes of double strength native sample buffer (20% glycerol, 0.01% bromophenol blue, 0.1 M Tris/HCl, pH 7.3).

Samples are electrophoresed as described in Figure 2 legend and visualized by negative staining without denaturation, based on the production of an insoluble dye complex which occurs only in the absence of protein (Zoion Biotech, Newton, MA). The excised bands are homogenized in 100-400 µl of 0.1M NaHCO³, 0.05% Triton X-100®*, 0.05% Tween-20®** (pH 7.0) at 3,000-4,000 RPM for 15-30s with an Eppendorf fitting pestle homogenizer, using a high speed mixer, (part numbers: KT749515-0000 or KT749520-0000, VWR pestle L-04368-02 Cole Palmer motor driven mixer). The emulsified slurry is placed into a sonicator bath at 22°C and prepared for recovery as described in homogenization protocol and legend of Figure 2 below, using Microcon-30 in combination with Micropure. Enzyme activity (Units/ml) is determined by continuous spectrophotometric rate assay, using the respective assays, conditions and absorbance wave lengths. See Table 1 and Figure 2.

SDS-Page
High purity grade urea, dithiothrietol (Bethesda Research Labs), methanol and acetonitrile (Becton Dickinson), trifluoracetic acid (Pierce), reduced Triton 100 (Aldrich), SDS and Coomassie blue R250 (Biorad), trypsin (Boehringer Mannheim) and endo lys C (Wako) are used with the highest grade reagents available. [125I]-BSA and [125I]-FSH (Dupont) are used with BSA, FSH, phosphorylase b, enolase and cytochrome c (Sigma). Extraction and digestion buffer solutions are filtered through a 0.22 µm filter (Nalgene). Gel electrophoresis is performed with the Tris-Tricine buffer system, using 0.5 mm thick gels of 10% acrylamide. The excised gel is placed into 100-400 µl of 100 mM NaHCO3, 8M urea, 3% SDS, 0.5% reduced Triton X-100 and 25 mM DTT (extraction buffer), then homogenized at 3,000-4,000 RPM in short bursts for 30-45 sec with an Eppendorf fitting pestle homogenizer, using a high speed mixer, (see above for details). The slurry is finally placed into a warm sonicator bath at 50-60°C for 1-3 hours or overnight without sonication.

To improve protein digestion, alkylation of sample can be performed either in the gel slurry before concentration or in the Microcon unit after the first concentration step. Iodoacetamide is added to a final concentration of 100 mM at 45°C for at least 30 min in extraction buffer (4). Proteins are electrophoresed in 10% mini SDS PAGE (0.5 mm thick), using the Tris Tricine buffer system (3). Gels are stained either with 0.1% Coomassie brilliant blue R250 (CBR250) in 50% methanol /10% acetic acid and destained in 7% acetic acid/12% methanol or preferably with a negative staining technique that permanently stains the gel without fixing the protein (Zoion Biotech). To remove CBR250, protein bands are placed into 1 ml of 50% methanol (wash buffer) into the Microcon vial and subjected to bath sonication at 50-60°C for 5-15 min or until gel becomes clear. Routinely, recoveries are greatly improved without fixing with the negative staining technique discussed in the results section.

Microcon/Micropure
A 0.22 µm Micropure separator is placed into a Microcon-10 concentrator (or Microcon-30, depending on the MW of the desired protein.) See Fig. 1. The 0.22 µm filter in the Micropure unit keeps gel from entering Microcon to contaminate the sample during centrifugation. To extract sample from gel, the gel is first homogenized to a slurry. After transferring the slurry to the Micropure unit, the homogenization vial is washed with an additional 100 µl of buffer which is transferred to Micropure, for a total volume of <350 µl. The Micropure/Microcon assembly is then centrifuged in a fixed-angle rotor at 13,000 × g for 20 min, concentrating the protein above the Microcon membrane below the Micropure unit. The latter is discarded. If further washing is necessary, 400-500 µl of wash buffer can be added to Microcon and spun for 30 min. For a buffer exchange, 400 µl of the desired buffer can be added and reconcentrated to 25-50 µl. For final sample collection, the Microcon unit is inverted into a collection tube and the sample recovered by 5-minute centrifugation at 5,000 × g.

Digestion and HPLC
Recovered samples are treated with 0.25-0.5 µg of either trypsin or endo lys c in 50-100 µl volumes of digestion buffer for 24h at 37¼°C. Following digestion, samples are either injected directly onto a column or acidified to pH 3 with TFA and stored at -20°C. Generated peptides are analyzed by reverse-phase HPLC with a MatrexTM C18 column (Amicon), using an acetonitrile gradient as described in Figure 3 legend.


Results

High-speed spins were shown to improve protein yields following extraction by sonication and incubation of protein-containing gels compared with passive diffusion. Examples of high recoveries by homogenization and centrifugation of native proteins from non-denaturing gels are shown in Table 1. These results demonstrate excellent enzyme activity recovery for native proteins glucose 6-phosphate dehydrogenase, ß-galactosidase and alkaline phosphatase, following gel electrophoresis. A negative staining technique allows for sample detection with minimal denaturation, followed by mild homogenization and extraction, using sonication in the presence of low amounts of detergents at 22S°C. In addition, continuous spectrophotometric rate determination assays for G6PDH and alkaline phosphatase demonstrated that 75-85% of the activity can be recovered, whereas less than 50% of ß-galactosidase activity was recovered under these conditions. The simultaneous removal of homogenized gel by Micropure and concentration/wash of sample followed by diafiltration permits protein extraction in the necessary detergents and buffers before transfer for assay of enzyme activity.

The recovery of enzyme activity can be achieved by homogenization, sonication, removal of gel and sample concentration with Micropure/Microcon (see Figure 2). Enzymatic activity improves with longer extraction times as shown by the increase in rate constants from slope 1 to slope 4. In addition, the presence of detergents with sonication for one hour at 25°C yielded the highest recovery shown in slope 2. Activity (units/ml), was determined by the maximum linear rate for both the test and blank samples where one unit can oxidize 1.0 µmole per minute of D-glucose 6-phosphate to 6-phospho-D-gluconate in the presence of ß-NADP at pH 7.4 and 25°C(6).

The efficiency of sample recovery following SDS-PAGE was demonstrated with labeled BSA and FSH using Micropure and Microcon. This was shown to be similar for the two proteins and ranged from 65 to 75% of total gel radioactivity after 2 hours of extraction with sonication. However, optimal recovery was achieved when overnight extraction was performed at 55°C.

The ability to digest gel-extracted proteins following Micropure / Microcon processing is shown in Figures 3 and 4 and described in the respective legends. HPLC-UV contaminants of gel blank (homogenized gel not containing protein removed by Micropure /Microcon-10) results in minimal UV contaminants, as shown in the lower left quadrant in Figure 3. The low level of UV contaminates is a function of the efficient sample diafiltration with Microcon. To achieve optimal digestion of gel extracted protein, reduction and alkylation was performed prior to extraction as described in the legends.


Conclusion

Micropure in combination with Microcon-10 or -30 can be used to perform sample recovery from polyacrylamide gels for a variety of applications. These include recovery of biologically active proteins from non-denatured gel, N-terminal sequencing of proteins and detailed structural analysis of generated peptides. The protocols presented can be appropriately modified to further improve sample recovery from gels 6 SDS, using a negative staining gel protocol. Without compromising detection limits, the negative staining procedure prevents sample-gel fixation and allows for efficient extraction and increased solubility of excised protein.

The Micropure/Microcon combination can simultaneously separate gel and extract protein during centrifugation with high efficiency and minimal sample loss. These devices facilitate extraction by maintaining sample solubility in a defined milieu that minimizes protein aggregation. In the case of maintaining activity of native proteins with non-denaturing gels, Microcon-30 is used to exchange detergent-containing extraction buffer with the appropriate assay buffer via diafiltration. Results demonstrate that recovery of enzymatic activity is sample dependent under variable extraction conditions. The ease of use with this device permits multiple samples for optimizing extraction conditions.

To obtain structural information from SDS-containing gels, extensive diafiltration eliminates most UV contaminants that interfere with high-sensitivity HPLC peptide mapping or sequencing. The results demonstrate that extracted protein must remain soluble throughout the procedure in order to achieve recoveries greater than 50%. The addition of detergents or chaotropes facilitates protein solubilization and extraction during homogenization and incubation. In addition to sonication during extraction, elevated temperatures improve the recovery of denatured proteins from SDS gels. This data demonstrates how the Microcon/Micropure unit in combination with non-denaturing gel electrophoresis, provides a powerful tool for purifying both native and denatured proteins for subsequent isolation and analysis.


References

  1. Aebersold, R. (1991). Advances in Electrophoresis (Chrambach, A., Dunn, M.J. and Radola, B.J. Eds), VCH Verlag Weinheim, Germany, Vol. 4, 81-168.

  2. Yuen, S.W., Chiu, A.H., Wilson, K.J. and Yuan, P.M. (1989) BioTech 7, 74-82.

  3. Schagger H.,von Jagow, G. (1987) Anal. Bioc. 166, 368-379.

  4. Riviere, L.R., Fleming, M., Elicone, C., Tempst, P. (1989) Techniques in Protein Chemistry (Villafranca, J.J., ed), Academic Press, New York, 171-190.

  5. Stone, K.L., McNulty, D.E., LoPresti, M.L., Crawford, J.M., DeAngelis, R., Williams, K.R. (1992) Techniques in Protein Chemistry (Angeletti, R.H. ed), Academic Press, New York, 23-34

  6. Noltman, E.A., Gubler, C.J., and Kuby, S.A. (1961) Journal of Biol. Chem 236, 1225-1230

Table 1. Rate determination of native proteins recovered from non-denaturing gels under a variety of conditions.
Native ProteinControl1 Hour
+
sonication		15 min
+
sonication		1 Hour
(U/ml)
Glucose 6-phosphate
dehydrogenase		19.5916.185.482.06
ß-Galactosidase37.2512.341.850.95
Alkaline phosphatase9.88.092.053.56


Figure 1. Combined Micropure separator and Microcon for gel extraction. Component view, center: Microcon at bottom, Micropure middle, cap at top. Left: Complete assembly, including vial at bottom. Vial at lower right is used to disperse the acrylamide gel by high-speed homogenization in extraction buffer with pestle.


Figure 2. Continuous spectrophotometric rate determinations from native, gel-extracted G6PDH. Approximately 3 µg of G6PDH was electrophoresed, excised using a negative (nonfixing) stain, and extracted as described in Methods. Assays were performed in 200 µl reaction cocktail mix of 50 mM glycylglycine, 2 mM D-glucose 6-phosphate, 0.67 mM ß-nicotinamide adenine dinucleotide phosphate, 10 mM magnesium chloride and 0.03-0.06 unit glucose-6-phosphate dehydrogenase 6. All samples assayed contained 20 µl of sample (in extraction buffer) and 180 µl reaction cocktail under the following conditions: Control G6PDH that was loaded onto gel (1), 1 hour sonication in extraction buffer containing 0.1M NaHC03, 0.05% Triton X-100 and 0.05% Tween 20 pH 7.0 (2), fifteen min sonication in extraction buffer (3), one hour extraction without sonication (4), blank containing reaction cocktail and 20 µl of extraction buffer (5).


Figure 3. HPLC separation of tryptic peptides. Separation from bovine serum albumin that had been run on SDS-PAGE, excised and homogenized as described in Methods (top left) and blank homogenized gel containing only 0.5 µg trypsin for control blank (lower left). Phosphorylase b gel that was homogenized and not alkylated / reduced (top right) or alkylated / reduced (lower right) as described in Methods section. Approximately 5 µg of sample was loaded onto each gel. Absorbance full scale was 0.05 at 214 nm.


Figure 4. HPLC separation of endo lys c peptides from enolase (left) and cytochrome c (right). Samples had been run on SDS-PAGE, excised, homogenized and prepared for HPLC as described in Methods. Lower chromatograms are from samples that were reduced and alkylated during homogenization, compared with extracted, unreduced samples (top).


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