Resources results for "NAC1"
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The PTMScan O-GlcNAc [GlcNAc-S/T] Motif Kit #95220 employs an antibody that will immunoprecipitate peptides with the native O-GlcNAc PTM on serine (S) or threonine (T) residues. To identify these peptides by mass spectrometry (MS), the fragmentation method most commonly employed is high energy collisional disassociation (HCD), or electron transfer dissociation (ETD). HCD fragmentation can produce the O-GlcNAc oxonium ion (+204.08 m/z); as well as oxonium ion fragments of m/z 186.07, 168.06 144.06, 138.05 and 126.05.
The ETD method of fragmentation has the advantage of not breaking the O-GlcNAc bond to the serine or threonine residue, improving the efficiency of localizing the PTM correctly on the parent peptide. See Ma and Hart Clinical Proteomics 2014, 11:8; http://www.clinicalproteomicsjournal.com/content/11/1/8.
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CIP/Lambda Phosphatase Treatment of Cell Lysates:
1) Wash cells 2X with ice-cold PBS.
2) Add cold 1X cell lysis buffer that does not include phosphatase inhibitors [1-2 E7 cells per ml or 0.4ml per 10 cm plate (80-90% confluent)]. Our cell lysis buffer is prepared as follows:
20 mM Tris-HCl (pH 7.5)
150 mM NaCl
1 mM Na2EDTA
1 mM EGTA
1% Triton
1 ug/ml leupeptin
1mM PMSF
3) Add 20ul Quick CIP (NEB #M0525; https://www.neb.com/products/m0525-quick-cip#Product%20Information) per 400ul of cell extract.
4) Incubate with Quick CIP at 37C for 1 hour.
5) Add 20ul lambda protein phosphatase (CST