General recommendations

It is extremely important to keep gel sample handling as clean as possible to prevent keratin and other protein contaminations, especially when dealing with low abundance proteins of interests. This means wearing gloves, a lab coat and a hairnet during the entire preparation process, using a clean apparatus for running gels etc., using siliconized polypropylene tubes as well as low-retention tips for transferring your samples and gel plugs.

Excise gel bands on extremely clean surfaces using new razor blades or scalpels. Ideally this should be done in a laminar flow hood (tissue culture type). When excising bands of interest cut as closely to the staining boundary as possible. If using 1-D gels, reduce the size of gel pieces to 1-2 mm in each dimension with a clean scalpel. Pieces that are too large will result in reduced peptide recovery. Pieces that are too small can be lost during pipetting steps. Most 2-D spots are of sufficient size when excised. All gel plugs picked up by robotic picker will be ready for the following steps.

Materials List

Code No	Name	Supplier	Description
A-6141	Ammoniumbicarbonate Ambic	Sigma	n/a
327190100	D,L 1,4 Dithiothreitol	Acros Organics	DNAse and RNAse and Protease free
122271000	Iodoacetamide	Acros Organics	n/a
270480010	Formic acid	Acros Oganics	n/a
T-6508	Trifluoroacetic acid	Sigma	n/a
V5111	Sequencing grade modified Trypsin	Promega	5 bottles a’ 20 µg

Protocol

A. Wash Gel Pieces

1. Add 100 µL H2O and let sit for 5 minutes, then remove and discard H2O
2. Add 100 µL (50:50) 100 mM Ambic pH 7.8 acetonitrile (ACN) and let sit for 10 minutes, then remove and discard 50% Ambic:ACN
3. Add 50 µL acetonitrile and let sit for 5 minutes, then remove and discard ACN. Gel pieces should appear shrunken and opaque.
4. Dry gel pieces in a vaccum centrifuge or ventilated fume hood for 5-10 minutes.

Skip to Section C if not performing reduction and alkylation.

B. Reduction and Alkylation (may be optional for protein ID)

1. Add 20 µL of 10 mM DTT in 100 mM Ambic and incubate 45 mins at 56°C
2. Allow tubes to cool to RT, then add 20 µL of 55 mM IDA in 100 mM Ambic and incubate in the dark for 30-60 mins.
3. Repeat Section A above to wash.

C. In-gel Digestion

1. Prepare trypsin solution on ice- 10 ng/µL trypsin in 4 °C 50 mM Ambic with 10% acetonitrile.
2. Rehydrate the gel pieces with 15 µL of the trypsin solution per tube/well, and incubate on ice for 15-30 mins.
3. Add 10 µL 50 mM Ambic to cover the gel pieces, and plate in a 30 °C water bath overnight.

D. Collect Extraction for LC/MS or MALDI

1. Add formic acid to each sample that final formic acid concentration is 1.0% to stop enzymatic reaction.
2. Remove and save supernatant in another labeled tube.
3. Add 30 µl of 50% acetonitrile with 5% formic acid (solution A), let sit for 45 minutes.
4. Sonicate for 5 minutes, then remove supernatant and add to the tube from step 2.
5. Repeat steps 3-4 one time.
6. Add 30µl of 90% acetonitrile with 5% formic acid (solution B) and let sit for 5 minutes.
7. Remove supernatant and add to the tube from step 2 (3 extractions in total).
8. Dry supernatant down to dryness using speed vacuum.

E. Collect Extraction only for MALDI

1. Add formic acid to each sample that final formic acid concentration is 1.0% to stop enzymatic reaction.
2. Remove and save supernatant in another labeled tube.
3. Add 30 µl of 50% acetonitrile with 0.2% TFA (solution C), let sit for 45 minutes.
4. Sonicate for 5 minutes, then remove supernatant and add to the tube from step 2.
5. Repeat steps 3-4 one time.
6. Add 30µl of 90% acetonitrile with 0.2% TFA (solution D) and let sit for 5 minutes.
7. Remove supernatant and add to the eppi from step 2 (3 extractions in total).
8. Dry supernatant down to dryness using speed vacuum.