MS sample preparation by centrifugation using OMIX C18 tips_Gel bands
Day 1
Cut bands, chop each one into 1mm3 cubes
Add 200ul 50mM AmBic /50% ACN
Mix with the gel pieces by vortexing
Incubate @ 37°C for 30’; discard the sup
Add another 200ul 50mM AmBic /50% ACN if the gel pieces are still blue
Destain more as needed; Usually 2x 30’ should be enough for single band
Remove 50mM AmBic / 50% ACN when the gel pieces are completely destained
Add 100ul ACN, vortex, let the tubes set at RT for 2’ or more
Remove ACN from all samples
Dehydrate in speed vac for 10’
Samples can be stored @ -20°C or proceed to trypsin digest
Day 2
Prepare trypsin working solution: 12.5ng/ul in 50mM AmBic (trypsin stock 100ng/ul in 1mM HCl)
Prepare tubes on ice
Add 12.5ng/ul trypsin to cover (15ul per tube for single band cut from 15-well gel; may need to add more for 10-well gel band)
Let samples swell for 45’ on ice
After swelling, add 50mM Ambic to cover gel pieces if necessary (~5ul for 15-well gel or 10ul for 10-well gel)
Move the tubes to 37°C incubator to digest overnight (6 hrs at least) Day 3 (The gel extraction steps here are different from what Kelly does)
Add 0.5% TFA (w/v, final concentration) to stop digestion • Dilute 20% TFA stock to 2% first • Add 1/3 of digestion vol. of 2% TFA to digestion mix • Mix FTA with gel pieces and incubate @ RT for 5’
Remove the digest solution to 0.65ml low-binding tubes; hold @ RT or 4°C
Extract with 30ul of 0.1% TFA @ RT for 45’
Remove extractant, combine with digest solution (total vol. will be ~50ul for each 15-well gel band)
Desalt sample using OMIX tips (C18, 10ul for gel band) by centrifugation
Tip wet: 2x 10ul of 50% ACN (v/v), 0.1% TFA (w/v)
Spin @ 1000rpm for 1’ (same speed and time for later spinning steps)
Tip wash: 2x 10ul of 0.1% TFA (w/v)
Sample binding: let the sample passes through the filter once (load 20ul at a time; may have to load multiple times for each sample)
Save the flow-through @ -20°C in case the binding is not complete
Sample wash: 5x 10ul of 0.1% TFA (w/v)
Elute sample by centrifugation
Pre-wash one set of low-binding tubes to hold elution (both E1 and E2) with 100% ACN
Elution 1: 20ul 40%ACN / 0.5% HAc Use clean collecting tube (pre-washed with 100% ACN) Let everything pass through
Elution 2: 20ul 80%ACN / 0.5% HAc Let everything pass through Collect E1 and E2 in the same tube
Put the tube containing E1+E2 in the speed vac and dry completely