In-Gel Digestion of Proteins Separated by Polyacrylamide Gel Electrophoresi

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In-Gel Digestion of Proteins Separated by Polyacrylamide Gel Electrophoresi

In-Gel Digestion of Proteins Separated by Polyacrylamide Gel Electrophoresis (According to EMBL Protein and Peptide Group, 1997)

1. Excision of protein bands (spots) from polyacrylamide gels
Rinse the gloves you use with water to avoid traces of dust in your sample.
Rinse the gel with water.
Excise spots with clean pipette tip (f 2 mm) cutting as close to the edge of the spot as possible (to reduce the volume of 'background' gel)
Transfer gel spot into a well of a 96-microtitre plate (Costar # 3363 + 3092)

2. Reduction and alkylation
This step is omitted because proteins are already reduced and alkylated during the equilibration after the 1st dimension. Include maybe for small protein amounts.

3. Washing of gel pieces (Coomassie, Sypro Ruby)
Washing to remove unpolymerized acrylamide (which may react with the peptides) and other (buffer) contaminants (which may give a higher background during MS measurement)
Wash the sample 5 min in 80 l 0.1M NH4HCO3
Add an equal volume of 100% Acetonitrile and vortex for 5 min
Spin down the gel particles (table-top centrifuge, max. 2800 rpm)
Remove the solution

Repeat this washing step twice.
Shrink the gel pieces with 150 l 100% Acetonitrile (HPLC grade) for about 5 min
Remove the solution
Dry down gel particles in a Speed Vac

4. In-Gel digestion with Trypsin
Precool tubes with the dried gel particles and the Trypsin solution on ice
Rehydrate the gel particles in the trypsin solution (12.5 ng/l Trypsin [Promega #V5111] in 50 mM NH4HCO3) for 30-45 minutes at 4°C. Add just enough solution (20 l) to cover the gel pieces (you only need to have the trypsin in the gel pieces to digest the proteins)
After 15 minutes, check out the samples and add more trypsin solution if the solution is completely absorbed by the gel pieces
After a total of 30-45 min rehydration, remove the remaining trypsin solution containing unnecessary Trypsin (to reduce the amount of self-digested trypsin peptides in the final sample)
Wash briefly with 80 l 50 mM NH4HCO3 (just add and remove) to remove the trypsin on the outside of the gel pieces
Add 80 l 50 mM NH4CO3 without Trypsin to have the gel pieces covered during the overnight digestion (16 - 20 h) at 37°C
Spin down condensed water droplets
Transfer liquid supernatant ('digest-solution') which can already contain small peptides (<2000Da) to a fresh 0.5 ml tube or a 96-tubes-plate

5. Extraction of peptides from the gel piece
Add 10-15l of 25 mM NH4HCO3 (maybe more) (to adjust the pH)
Incubate for 15 min at 37°C while shaking (Vortex)
Spin down and add 100% Acetonitrile (1-2 times equal the volume of the gel, 80 l)
Incubate for 15 min at 37°C while shaking
Sonicate for 5 min in sonication bath at 37°C (17L )
Spin down liquid and transfer the 'basic extracted' peptide containing solution to the same tube as the 'digest solution'
Add 40-50 l of 5% formic acid (to adjust the pH)
Incubate for 15 min at 37°C while shaking
Spin down and add 100 % Acetonitrile (1-2 times equal the volume of the gel, 80l)
Incubate for 15 min at 37°C while shaking
Sonicate for 5 min in sonication bath at 37°C
Spin down and transfer the 'acid extracted' peptide containing solution to the same tube, together with the basic extracted peptides and the 'digest solution'
Freeze in liquid nitrogen, then dry in a Speed-vac.
At this point, samples can be stored at -20°C.

6. Mass spectrometry
Dissolve the sample in ~ 10 l 0.1% TFA
Inject ~ 5 l in the LCQ Deca from Finnigan

7. Reduction and alkylation
Wash the particles with 100-150 l of water (5min), spin down and remove the liquid.
Add acetonitrile (3-4 gel volumes) and wait for 10-15 min, spin particles down and remove all liquid, dry down in a speed vac.
Swell the gel pieces in 10 mM DTE/ 0.1 M NH4HCO3, enough to cover the gel, and incubate for 30 min at 56°C (waterbath) to reduce the protein.
Spin down and remove excess liquid, shrink 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 l 0.1 M NH4HCO3 for 15 min.
Spin down, remove all liquid and shrink with acetonitrile.
Remove all liquid and dry gel particles down in a speed vac.

Link: http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=2514