Hello,
I want to do western blotting with Jurkat cells. Up to now I did western blotting with solid tumors by using RIPA 0.5-1.0 buffer and 15 minutes of centrifugation at 14.000g. I used the same procedure on Jurkta cells. I have measured very little protein concentration in BCA assay. I wonder what should I change while working with Jurkats. In one article, I saw they need light centrifugation. Any more suggestions??
Hello
in paper by "Aitor G et al " , cells were harvested by centrifugation, washed twice with PBS, and resuspended in 500 l of buffer A (10 mM HEPES (pH 7.6), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.75 mM spermidine, 0.15 mM spermine, 1 mM DTT, 0.5 mM PMSF, 10 mM Na2MoO4, and 2 g/ml each of inhibitors leupeptin, aprotinin,and pepstatin A). After 15 min at 4°C, 5 l of a 10% Nonidet P-40 solution was added. Samples were vortexed for 10 s and centrifuged for 20 min at 3000 rpm and 4°C. The supernatants were used as cytosolic extracts. To avoid cytosolic contamination, nuclei were washed twice with 200 l of buffer A. For nuclear protein extraction, 50 l of buffer C (20 mM HEPES (pH 7.6), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT,0.5 mM PMSF, 10 mM Na2MoO4, and 2 g/ml each of inhibitors leupeptin, aprotinin , and pepstatin A) was added, and nuclear pellets were incubated for 30 min at 4°C with gentle agitation. Samples were centrifuged for
10 min at 14,000 rpm and 4°C, and supernatants were used as nuclear
extracts. Whole-cell protein extracts from wt Jurkat cells were prepared, as
previously described (37), using radioimmunoprecipitation assay (RIPA)
buffer, containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet
P-40, and 0.25% Na-deoxycholate, and supplemented with protease inhibitor
mixture tablets (Roche). In any case, protein concentration was determined
by the bicinchoninic acid (BCA) spectrophotometric method
(Pierce).
see full text in links here .
Hope help you . Good Luck
http://www.bosterbio.com/protocol-and-troubleshooting/western-blot-principle
http://www.jimmunol.org/content/180/4/2429.full.pdf
I have been working with Jurkat cells for a number of years. A protocol for Western blotting can be found in,
J Leukoc Biol. 2000 Aug;68(2):293-300.
Chronic PMA treatment of Jurkat T lymphocytes results in decreased protein tyrosine phosphorylation and inhibition of CD3- but not Ti-dependent antibody-triggered Ca2+ signaling.
Ahnadi CE, Giguère P, Gravel S, Gagné D, Goulet AC, Fülöp T Jr, Payet MD, Dupuis G.
I hope this is helpful to you.
G. Dupuis
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