Analysis of Proteins using Small Format 2D Gel Electrophoresis
Preparation of protein samples
Intracellular virus proteins
The following method has been developed principally for the analysis of intracellular proteins from picornavirus infected HEp-2C cell monolayers. Modifications to the incubation times can be made to use the protocol with other virus-host cell systems.
Between 2.2 x 10^7 and 7.1 x 10^8 TCID50 of virus are inoculated onto confluent HEp-2C cell monolayers prepared in 30mm petri dishes. After a 30 minute adsorption at room temperature the cells are overlaid with 2ml of MEM containing 2% newborn calf serum (NBCS). The uninfected and virus infected HEp-2C cell monolayers are incubated for 4-5 hours at 37C before being washed twice with PBS-A. The cell monolayers are then overlaid with 0.5ml of methionine-free MEM (without NBCS) and incubated at 37C for 30 minutes at which time L-[35S]-methionine (final concentration 40µCi per ml) is added and the incubation continued for a further 30 minutes at 37C. The cell monolayers are then washed twice with PBS-A and lysed in 0.2 ml of 2D lysis buffer (0.01M Tris-HCl, pH 7.4, 1mM EDTA, 8M urea, 0.05M dithiothreitol, 10% (v/v) glycerol, 5% (v/v) Nonidet P-40, 200µg/ml RNAse A, 6% (w/v) pH 3.5-10 carrier ampholytes ("Resolyte", Merck Ltd.)) for 5 minutes on ice. The nuclei are removed by centrifugation at 11,500 x g for 5 minutes and the supernatant stored at -20C for later analysis by gel electrophoresis. The incorporation of radioactive methionine into the proteins is determined by TCA precipitation - this provides a means of balancing the sample loading to the 2D gels. Routinely between 40,000 and 200,000 TCA-precipitable counts in a maximum volume of 10µl are loaded per gel.
Further information is also available on the preparation of cell proteins for 2DE.
Bacterial proteins
Cellular proteins from the majority of gram negative bacteria can be processed using the following one-step cell lysis method. In all cases the bacteria are grown overnight on the appropriate agar culture plates.
Single bacterial colonies are inoculated onto agar plates and grown overnight at 37C to produce heavy bacterial growths. The bacteria are removed from the surface of the agar using a sterile swab which is immersed into 0.5ml of 2D lysis buffer (0.01M Tris-HCl, pH 7.4, 1mM EDTA, 8M urea, 0.05M dithiothreitol, 10% (v/v) glycerol, 5% (v/v) Nonidet P-40, 6% (w/v) pH 3.5-10 carrier ampholytes ("Resolyte", Merck-BDH)) in microfuge tubes; the bacteria can be dislodged from the swab by rubbing the swab against the side of the tube. The bacteria lyse within 1 or 2 minutes. Large cell debris are removed by centrifugation at 11,500 x g for 5 minutes. The supernatant is stored at -70C until required for analysis. The protein concentration of the sample can be determined using a standard protein assay kit. Depending on the assay system, the lysis buffer used to prepare the bacterial proteins may give a background equivalent to approximately 5mg/ml of protein as determined from a standard curve; this compares with a protein concentration of 10-16mg/ml of protein for the bacterial lysates (i.e. including the lysis buffer). For the analysis of the bacterial proteins by 2D-PAGE, the equivalent of 70µgm of bacterial protein in a maximum volume of 10µl is loaded to each first dimension gel.
Further information is also available on the preparation of bacterial proteins for 2DE.
Protein analysis on small format 2D gels
The analytical system presented below was developed using the Mini Protean II system from Bio Rad Laboratories for the first dimension gels and the Mighty Small slab gel system (Pharmacia Biotech) for the second dimension.
First dimension
The first dimension tube gels are cast in 75 mm (1 mm internal diameter) capillary tubes and consist of 5% (w/v) acrylamide (0.25% (w/v) N,N''-methylenebisacrylamide) 8M urea, 2% (v/v) Nonidet P-40 and 2.5% (w/v) pH 4-8 carrier ampholytes ("Resolyte", Merk Ltd.). The acrylamide solution containing urea and NP40 is treated with a mixed bed ion exchange resin and stored at 4C for up to one month; the ampholytes are added to the gel solution immediately before use. If required, the gel solution can be degassed prior to the addition of the catalysts (ammonium persulphate and TEMED). The gel is left to polymerise overnight with an overlay of water-saturated butanol. The tube gels are assembled in the first dimension gel apparatus as described by the manufacturer using 0.1M NaOH as the catholyte and 25mM H3PO4 as the anolyte. Proteins are loaded at the cathode on the first dimension gel and electrophoresed at 500 volts for 15 minutes and then 1500 volts for 2 hours. The electrophoresis times can be determined empirically for specific samples.
NEPHGE can be used in the first dimension to identify basic proteins. For these analyses the first dimension gels are modified as follows. The pH 4-8 carrier ampholytes are replaced with 2.5% (w/v) pH 3.5-10 carrier ampholytes and the protein samples are loaded at the anode. The gels are electrophoresed at 1500 volts for 1 hour.
Second dimension
Following electrophoresis in the first dimension, the tube gels are expelled onto the surface of the second dimension slab gels and equilibrated in buffer (0.125M Tris-HCl, pH 6.7, 2.5 mM dithiothreitol, 2.3% (w/v) SDS) for 7 minutes before embedding in 1% (w/v) agarose prepared in 0.125M Tris-HCl, pH 6.7, 0.1% (w/v) SDS with bromophenol blue to colour.
The second dimension slab gels (70 x 80 mm) are prepared in batches of 12 gels using a multi-gel casting system. A standard Tris-Glycine electrophoresis buffer system is used containing 0.1% (w/v) SDS. The gels are prepared using a 7-15% linear polyacrylamide gradient which is stabilised with a 5.6-75% glycerol gradient during preparation; a stacking gel is not required. The slab gels are electrophoresed at 200 volts for 2 hours by which time the bromophenol blue dye reaches the end of the gel. The gels are cooled with running tap water during the electrophoretic run.
Protein detection methods
The method used to locate the proteins following 2D-PAGE depends on the nature of the original sample.
Using the radiolabelling protocol described above, the incorporation of the radioactive methionine is sufficient to detect the proteins by direct autoradiography with a 4-5 day exposure - it is not normally necessary to use an amplification system (e.g. DMSO and PPO). The lowest incorporation is found for the virus-infected cell lysates.
Non-radioactive bacterial proteins are detected by staining with Coomassie brilliant blue by one of two methods. Proteins are stained overnight with 0.2% (w/v) Coomassie brilliant blue R250 in 50% (v/v) methanol, 5% (v/v) acetic acid; the gels are then destained in the same solution without the stain. Alternatively, the proteins are stained using the colloidal staining method described by Anderson et al. [Electrophoresis (1991) 12, 907-930] and modified as follows to take into account the small gel format. The gels are processed in batches of 10 and fixed overnight in 200 ml of 50% (v/v) ethanol, 2% (v/v) phosphoric acid. The gels are then washed three times for 30-60 minutes in a minimum of 500 ml tap water and equilibrated for 60 minutes in 200 ml of 34% (v/v) methanol, 17% (w/v) ammonium sulphate, 2% (v/v) phosphoric acid before adding 0.2 g Coomassie brilliant blue G250. The gels are continuously agitated in the stain for at least 4 days to allow maximum staining.
Gels to be processed for autoradiography are washed in water and dried under vacuum onto blotting paper using a heated gel dryer. Stained gels, not to be processed for autoradiography, are washed in excess tap water and then soaked in 0.5% (v/v) glycerol for 60 minutes prior to being dried between cellophane sheets in a heated air dryer.
Analysis of 2D proteins profiles
Images of the gels to be analysed are captured using a various methods. Many of the images presented on this site were captured as 1024 x 1024 pixel images using a video camera attached to a Gemini image analysis system (Applied Imaging). More recently images are captured using either a CCD camera (Astrocam Ltd.) as 1536 x 1024 pixel images (8-bit or 16-bit images) or Molecular Dynamics Personal Densitometer. In all cases computer assisted analyses of the 2D protein profiles use Phoretix-2D (Phoretix International) and all spot detection and matching processes use the routines available from within this software. Further details of the matching of the images with each other can be obtained from the discussion of the Haemophilus 2D Database structure.
Where stated, the isoelectric points of the proteins are determined by co-electrophoresis with carbamylated creatine kinase (Merk Ltd.). The apparent molecular weights of the proteins are determined by co-electrophoresis with the following proteins of known molecular weights (obtained from Pharmacia Biotech): bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa) and trypsin inhibitor (20.1 kDa).
Post-electrophoretic identification of proteins
Various protocols can be used for the identification of proteins following the analysis of cell lysates by 2DE. Many of the analyses described at this site have used bacteria for which complete genome sequences have been determined. This simplifies the identification of the proteins synthesised by these organisms. Protein identifications can be made for related strains/isolates based on homology.
Protein identifications have been achieved using peptide mass mapping following the protocols described by Shevchenko et al. (1996) and Wilm et al. (1996). Further details on the methods used in Aberdeen are available from Cash et al. (1999). These procedures use in-gel trypsin digestion for the proteolytic step. Peptides are sized on a PerSeptives Biosystems Voyager-DE STR MALDI-TOF mass spectrometer and used to search a non-redundant protein sequence database maintained at EMBL and EBI.
