This is a protocol we have adapted from Wolfgang H. Fischer at the Salk Institute in La Jolla, California. It works virtually every time. He has sugggested that there should be 5-50 picomoles of protein (if possible) and if doing 32P labeled proteins then at least 10,000 counts.
For a gel slice 3 x 10 mm.....
wash 15 min with 10 mM mercaptoethanol (200 uL)
wash 2 x, 15 min 40% n-propanol (200 L)
wash 2 x, 15 min 50% acetonitrile/50mM triethylammonium bicarbonate (200 uL)
wash once with 50 mM triethylammonium bicarbonate (200 uL) for 5 min
add 150 uL of 50 mM triethylammonium bicarbonate and 8 uL of 1 ug/25 uL Asp-N (Boehringer-Mannheim)
incubate at 37deg C overnight
At this point one can shoot this directly onto the microbore HPLC or we have had some success with combining the digest with a wash of the gel slice with 200 uL 50% aceonitrile/0.1%Trifluoracetic acid and evaporating the combined aliquots to a small volume.
Good luck!
--------------------------------------------------------
Gordon Alton, PhD
Gene and Protein Discovery Group
Signal Pharmaceuticals Inc.
5555 Oberlin Drive
San Diego, CA 92121
Email: galton@signalpharm.com
Phone: 619-558-7500 x8252
Fax: 619-623-0870
WWW: http://www.signalpharm.com
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-----Original Message-----
From: Kathryn Sunn [SMTP:k.sunn@garvan.unsw.edu.au]
Sent: Sunday, August 01, 1999 10:24 PM
To: Recipients of ABRF List
Subject: ASP-N digests
Dear All
Does anyone have a good protocol for ASP-N digests in gel slices?
Thanks in advance
Kate
___________________________________________________________________
Kathryn Sunn BSc (Hons)
Postgraduate Student
Bone and Mineral Research Program
Garvan Institute of Medical Research
384 Victoria St, Darlinghurst
St Vincent's Hospital Tel: (02) 9295 8260
Sydney NSW 2010 Fax: (02) 9295 8241
Australia email: k.sunn@garvan.unsw.edu.au
___________________________________________________________________