Measures to take to avoid contamination with keratin
All procedures including gel band excision to be carried out in a dust-free area or laminar flow hood.
Wear powder-free nitrile gloves rinsed with water and changed often.
Prior to a new digestion prepare fresh triethylammonium bicarbonate (TEAB) in glass universals rinsed with fresh ultra-pure 18MW Decant fresh acetonitrile into a rinsed universal.
Clean glassware/scalpel blades with methanol.
Use fresh microfuge tubes from a sealed bag. Open microfuge tube lids carefully, avoid flicking open.
Replace lids on all solutions straight after use to prevent contamination with keratin.
Care when preparing new trypsin stock solutions as keratin contamination here will affect all subsequent digests.
100mM TEAB – Add 1ml of 1M TEAB to 9ml ultra-pure 18MW water and vortex gently to mix.
50mM TEAB – Mix equal volumes of 100mM TEAB with ultra-pure 18MW water and vortex gently to mix.
10mM DTT – Make 100mM stock; weigh out 15mg of DTT into a centrifuge tube and add 1ml of 100mM TEAB. Dilute 10-fold for working solution (100ml 10mM DTT and 900ml 100mM TEAB)
55mM IAM – Weigh out 10mg of IAM into a centrifuge tube and add 1ml 100mM TEAB. Light sensitive solution, make fresh immediately before use.
Trypsin - Make 0.1% TFA fresh in rinsed universal (5ml water, 5ul TFA]. Add 250ml of 0.1% TFA, vortex gently with lid on to ensure solubilisation. Divide into 8x 30ml aliquots in microfuge tubes and freeze at -20°C for future use.
Tryptic digestion
Excise bands of interest cutting as close to the edge of the band as possible. Chop into ~2 mm² pieces and transfer into a centrifuge tube. NB. Store gel pieces at 4°C in water until required.
Wash the gel cubes with 100mM TEAB (decant off water first if gel pieces have been stored) and decant. Volumes of TEAB and ACN (steps 2 to 8) are added in excess to cover the gel pieces.
Add acetonitrile, decant after one round, and add same volume again to dehydrate the gel pieces, decant and dry in speed vac for 5 mins fully and quickly. Whilst samples are drying prepare DTT solution.
Rehydrate the gel with 10mM DTT and heat at 56°C for 30 mins.
Decant DTT, add ACN (2x volumes; see step 3), dehydrate and dry in speed vac (5 mins). Whilst samples are drying prepare 55mM IAM solution.
Add 55mM IAM, incubate at ambient temperature for 20 mins in the dark.
Discard the supernatant, wash briefly with 100mM TEAB buffer then replace and wash for a further 5 mins and discard the buffer (go to step 9 if destaining not required).
If not completely destained at this stage (if using colloidal Coomassie stain) wash with 1:1 solution of TEAB:ACN in excess. Incubate at 37°C shaking for 30 mins (repeat this step until destained).
Decant the liquid, dehydrate once again with acetonitrile as in step 3 and dry off in a speed vac for 10 mins
Take trypsin aliquot/s (30ml) and add 200ml of 50mM TEAB to give a final trypsin concentration of 13ng/m Rehydrate the gel pieces in a minimal volume of trypsin solution (i.e., enough to cover and rehydrate the gel pieces) at 4°C for 20mins. Remove unabsorbed trypsin and add the same volume of 50mM TEAB to cover the gel pieces and keep them wet during enzyme cleavage. Incubate at 37°C for 2 hours then overnight at room temperature.
Peptide extraction
Decant supernatant from gel pieces and collect into a new centrifuge tube.
Wash gel pieces with 50mM TEAB for 5 mins at 37°C, decant and pool into tube from step 1. Use a minimal volume that will immerse the gel pieces.
Dehydrate gel pieces with ACN for 5 mins at 37°C, decant and pool supernatant into tube from step 1.
Repeat steps 2 and 3.
Freeze the peptide extract and dry down the pooled supernatants to completion in a speed vac; avoid over drying. Store at -80°C until required.
