Bioanalytical Research

Bioanalytical Research Group

Gel Electrophoresis

1. Excision of protein bands (spots) from polyacrylamide gels

Rinse the gel with water. Excise bands of interest with clean scalpel cutting as close to the edge of the band as possible. It is important to reduce the volume of " background " gel. Chop the excised bands into cubes (ca. 1x1mm2) . Transfer gel particles into a micro-centrifuge tube (0.5 ml or 1.5 ml Eppendorf). Do not touch gel with fingers ! Always wear gloves . Rinse gloves with water to wash out talcum powder and traces of dust.

2. Reduction and alkylation 

Wash the gel particles with 100-150 µl of water ( 5 min.) . Spin down and remove the liquid . Add acetonitrile ( ca. 3-4 times equal the volume of gel pieces ) and wait for 10-15 min until the gel pieces shrunk - they become white and stick together . Spin the gel particles down and remove all liquid . Dry down gel particles in a vacuum centrifuge . Swell the gel pieces in 10 mM dithiotreitol/0.1 M NH4HCO3 ( add the liquid enough to cover gel ) and incubate for 30 min. at 56°C to reduce the protein . In-gel reduction is recommended even if proteins were reduced prior to an electrophoresis . Spin down the gel particles and remove excess liquid . Shrink the gel pieces with acetonitrile . Replace acetonitrile with 55 mM iodoacetamide/0.1 M NH4HCO3 . Incubate for 20 min. at room temperature in the dark. Remove iodoacetamide solution . Wash the gel particles with 150-200 µl of 0.1 M NH4HCO3 for 15 min. Spin down the gel particles and remove all liquid . Shrink the gel pieces with acetonitrile. Spin down the gel particles and remove all liquid . Dry down gel particles in a vacuum centrifuge .

3. Washing of gel pieces ( for Coomassie-stained gels only )

If the gel pieces excised from the gel are still blue rehydrate them in 100-150 µl 0.1 M NH4HCO3 and after 10-15 min. add an equal volume of acetonitrile (to get 0.1 M NH4HCO3 /acetonitrile 1:1 v/v ) . Vortex the tube for 15-20 min. Then spin down the gel particles and remove all liquid . Shrink the gel pieces with acetonitrile . Remove all liquid. Dry down gel particles in a vacuum centrifuge . Repeat step 3 if necessary .

4. In-gel digestion with trypsin.

Rehydrate gel particles in the digestion buffer containing 50 mM NH4HCO3 , 5 mM CaCl2 and 12.5 ng/µl of trypsin at 4°C (ice bucket ) for 30-45 min. (After 15-20 min check out the samples and add more buffer if all liquid is absorbed by the gel pieces). Remove the remaining supernatant. Add 5-25 µl of the same buffer but without trypsin to cover gel pieces and keep them wet during enzyme cleavage . Leave the samples at 37°C overnight .

5. MALDI analysis of the supernatant after in-gel digestion.

After overnight incubation spin down the water droplets condensed on the tube lid. Take up small (1-2 µl) aliquot of the supernatant for MALDI analysis.

6. Extraction of peptides.

If Nano ES MS/MS analysis is necessary tryptic peptides should be extracted from the gel particles. Add 10-15 µl of 25 mM NH4HCO3 .Incubate at 37°C for 15 min with shaking . Spin down the gel particles and add acetonitrile ( 1-2 times equal the volume of gel particles). Incubate at 37°C for 15 min with shaking. Spin down the liquid and collect the supernatant. Add 40-50 µl of 5% formic acid .Vortex for 15 min. at 37°C . Spin down the gel particles and add acetonitrile ( 1-2 times equal the volume of gel particles). Incubate at 37°C for 15 min with shaking . Spin down the gel particles and pull the extracts together. Dry down the extracts in a vacuum centrifuge .