Gel Electrophoresis of Proteins
Peptide mapping involves controlled cleavage of a pure protein with small amounts of a pure protease to generate peptides of characteristic, reproducible sizes. These peptides can be separated on PAGE to produce a “fingerprint” characteristic of the protein. Peptide mapping can map cleavage sites in an unknown protein, or it can identify an unknown protein based upon its fingerprint identity with a previously tested sample.
The polyacrylamide gel used can be either denaturing or non-denaturing, but SDS PAGE is most often used because it gives molecular weight information about the peptides produced. Small amounts of protease are used, so that minor variations in time and temperature of incubations will not overly perturb the results.
Proteins for peptide mapping can be taken from bands sliced out of electrophoresis gels, or purified by standard means. Protocols are provided for the mapping of a pure protein and a protein in an acrylamide gel.
Peptide Mapping – Purified Protein
- Dissolve protein to 0.5mg/ml in digestion buffer (composed of 0.125M Tris HCl pH 6.8, 0.5% SDS, 10% glycerol and 0.0001 % bromopohenol blue) and heat to 100°C for 2 minutes.
- Cool to 37°C and digest with protease for 30 minutes (see table below for enzymes and amounts).
- Stop digestion by adding SDS to 2% (1/10 vol of 20% SDS) and 2-Mercaptoethanol to 10% and heating to 100°C for 2 minutes.
- Load 10 – 15 – 20 µl (5 -10 µg) on a 10 – 15% SDS PAGE gel for analysis.
Peptide Mapping – Protein in a Gel Slice
- Stain and destain as quickly as possible to avoid acid hydrolysis artifacts.
- Cut out band (containing 1-10µg of protein) of interest and rinse slice in cold deionized water. Cut slice to the width of a well, as it will be loaded into the analytical gel.
- Soak slice in 1X >stacking gel buffer and 1 mM EDTA.
- Place slice at the bottom of a well in the analytical gel (use a spatula or loading tip to place slice).
NB: The analytical gel for this protocol must have a stacking gel of at least 3 cm, to allow a space for digestion to occur.
- Overlay slice with stacking gel buffer + 20% glycerol.
- Overlay this with 10 ml of stacking gel buffer + 10% glycerol + protease + 0.01% Bromophenol Blue
- Run samples into stacking gel. When the tracking dye reaches the bottom of the stacking gel, turn off voltage for 30 minutes to allow digestion to occur. Then continue run as usual.
Note that smaller amounts of radiolabeled protein can be analyzed by this method.
Analytical gels for peptide mapping
The choice of gel system for the analysis of peptide mapping is dictated by the anticipated results. If a wide range of peptide sizes is anticipated, a< gradient gel may be required. For peptides over 7kd, standard Tris Glycine SDS PAGE gels will give superior results. Small peptides will require strongly denaturing fixatives to avoid loss of signal during staining. For extremely small peptides, analysis on native PAGE gelsmay be superior. In native protein PAGE, separation is based partly on charge to mass ratio. This can enable the resolution of peptides that would run too close to the SDS/dye front in SDS PAGE.
NEXT TOPIC: Native Protein Electrophoresis
- Sample Preparation for SDS-PAGE
- Sample Preparation for Native Protein Electrophoresis
- Peptide Mapping
- Native Protein Electrophoresis
- Measuring Molecular Weight with SDS-PAGE
- Isoelectric Focusing
- Immuno-Electrophoresis / Immuno-Diffusion
- Gel Preparation for Native Protein Electrophoresis
- Gel Electrophoresis of RNA & Post Electrophoretic Analysis
- Denaturing Protein Electrophoresis: SDS-PAGE
- Casting Gradient Gels
- Activity Stains
- Sample Preparation
- Electrophoresis Buffers
- Blotting and Detection Buffers
- Gel Matrices
- Gel Accessories
- Ultra Pure Reagents