a protocol we have used in our lab for five years now with good success is
as follows:
1. The digest is acidified to 2% TFA and the supernatant removed and reserved.
2. The gel slice is covered with 0.1% TFA (usually 50 to 70 microlitres for
a single gel slice) and extracted for 30 minutes. The supernatant is
removed and added to the reserved supernatant from step 1.
3. The extraction is repeated as per step 2, once with 30%
acetonitrile/0.1% TFA and once with 60% acetonitrile/0.1% TFA.
4. The combined supernatants are "SpeediVac'd" down to less than 50
microlitres prior to reversed phase HPLC
Extractions are performed by floating the tubes in a foam rack on a
sonicating waterbath, this method of extraction is chosen as a convenience
thing really, there is a sonicating waterbath in the instrument room
adjacent the lab, and it works well.
As for any protocol being "the best", I think you will get plenty of
alternatives suggested from many other experienced investigators in the
ABRF, there is even one one the ABRF home page (http://www.abrf.org) under
the Internal Protein Sequence Research Committee.
One slight advantage I have in my extractions is that I am able to
precisely monitor peptide recoveries since many of the phosphoproteins I
deal with are radiolabelled. In my experience, the above extraction
protocol, after a good digest, can yield greater than 98% recovery on a
wide range of peptides from short, hydrophilic ones to 30 residue,
myristoylated (very hydrophobic) ones.
Hope this helps....Ken
>Hi all:
>
>Does anyone know what the best way is to optimize the extraction of
>trypsin-generated peptides from SDS-PAGE gels? There are many methods out
>there that use solvents such as formic acid/water/isopropanol or
>acetonitrile/ammonium bicarbonate or just formic acid with or without
>sonication, etc. Anyone know which one works best? Thanks in advance!
>
>Regards,
>
>Robert Becklin
>University of Tennessee, Memphis
>956 Court Ave., Room A218
>(901) 448-5488
>rbecklin@am.utmem.edu
********************************
Ken I. Mitchelhill
The John Holt Protein Structure Laboratory
St. Vincent's Institute of Medical Research
41 Victoria Parade
Fitzroy 3065 Victoria
AUSTRALIA
Telephone: 61-3-9288 2480
Facsimile: 61-3-9416 2676
Email: k.mitchelhill@medicine.unimelb.edu.au
Laboratory: http://www.medstv.unimelb.edu.au/WWWDOCS/SVIMRdocs/JHPSL.html
ABRF: http://www.abrf.org
***********************************