BAF_Protocol_001 In-gel Digestion
Nicholas Sherman
Abstract
This protocol is for in-gel digestion of proteins, including large mixtures, to produce peptides for mass spectrometry analysis. The gel method can be useful for getting rid of detergents or small molecules that might interfere with minimal loss. The input is a gel band up to 1cm x 1cm. The output is a relatively clean peptide digest that is ready for quick cleanup by C18 tip.
Before start
REAGENTS : (All reagents to be prepared fresh for each digestion)
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Destain solution: 10 mL MeOH (methanol), 10 mL H2O (water), 500 μL Acetic Acid (glacial)
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100 mM ABC (ammonium bicarbonate): 0.158 g in 20 mL distilled H2O
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50 mM ABC: 0.079g in 20 mL distilled water
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ACN (Acetonitrile)
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10mM DTT: 0.0015 g in 1 mL of 100 mM ABC (Use screw caps microtubes. DO NOT mix until directly before you are ready to use)
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50 mM Iodoacetamide: 0.01 g in 1 mL of 100 mM ABC (Use screw caps microtubes. DO NOT mix until directly before you are ready to use)
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Trypsin solution: Keep on ice. Promega sequencing grade trypsin (cat. # V5113 (porcine)) dilute when ready to use in 50 mM ABC. It comes frozen in 40µL of 50 mM Acetic Acid
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Extraction solution 1: 5% formic acid in H2O (950/50)
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Extraction solution 2: 5% formic acid in 50% MeCN/H2O (500/450/50)
Steps
DAY 1:
Cut gel band into small pieces (~2 mm).
Add the pieces in a pre-cleaned 1.5 mL polypropylene tube.
Destain the pieces in 0.5 mL destain solution for ~2hrs.
Remove destain (using 1 gel load tip for all tubes in batch) and replace with 0.5 mL destain solution for 30min. It’s okay if some stain remains. It will be removed during further wash steps.
Remove the destain solution and dehydrate gel slices in 200 μL ACN (Acetonitrile) for 5 min.
Remove ACN and repeat.
Reduce the gel pieces in 30 μL of 10 mM DTT (dithiothreitol) for 0.5 h at room temperature.
Remove the DTT solution.
Alkylate in 30 μL 50 mM IA (iodoacetamide) at room temperature, in dark for 0.5 h. (get ice for 50 mM ABC).
Remove the IA solution.
Wash gel pieces with 100 μL 100 mM ABC (ammonium bicarbonate) for 10 min and remove.
Dehydrate gel pieces in 200 μL ACN for approximately 5 min. Remove ACN.
Rehydrate gel pieces in 200 μL 100 mM ABC for 10 min.
Remove ABC supernatant.
Dehydrate gel slices in 200 μL ACN approximately 5 min. Remove ACN and repeat.
Prepare trypsin.
For Gel Pieces: Add 960 μL of cold 50 mM ABC to 20 ugs of Promega trypsin (Part number V5113) (it comes diluted in 40 µL of 50 mM acetic acid) on ice (20ug/mL).
Add 30-50 μL of the trypsin solution to cover the gel pieces and rehydrate on ice for 30 min.
Microfuge and remove any excess trypsin solution and add 5-20 μL of 50 mM ABC. React overnight at 37°C.
Day 2:
Prepare Extraction Solutions:
a. Extraction Solution 1: H2O/Formic Acid (950/50)
b. Extraction Solution 2: ACN/H2O/Formic Acid (500/450/50)
DO NOT remove excess ABC supernatant from yesterday!!!
Add 10 μL Extraction Solution 1 to each tube (or more if needed to cover the gel pieces) Let rest for 10 minutes. Take off supernatant to a clean 0.5 mL tube.
Add 10 μL Extraction Solution 2. Incubate for 10 min. Take off supernatant to the same 0.5 mL tube.
Repeat the 10 µL Extraction Solution procedure. Take off supernatant to the same 0.5 mL tube.
Evaporate the sample via speed vac and reconstitute to 10-100 μL total volume (depending on the amount of total protein) with 0.1% Formic acid.
Perform desalting using C18 tips (BAF_Protocol_003):
– 10 µL tips for a small amount of proteins
– 100 µL for a higher amount of proteins.
