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Dear Tech Support Team,
Our customer intends to use ab69314 in IP and she wonders in regards to the protocol details. Is it possible to receive some protocol? Do you recommend to use wash buffer firstly with low pH and then with high pH in order to wash unbound ab from the beads?
Thank you for the assistance.
Asked on Sep 02 2013
The exact protocol will vary and depends on the expression level of the GFP fusion and the type of protein and the purpose of the experiment.
The full protocol can be found in Megan C. Yap*#, Morris A. Kostiuk*, Dale D. O. Martin, Maneka A. Perinpanayagam, Pieter G. Hak, Anjaiah Siddam, Janaki R. Majjigapu, Gurram Rajaiah, Bernd O. Keller, Jennifer A. Prescher, Peng Wu, Carolyn R. Bertozzi, John R. Falck and Luc G. Berthiaume (2010) “Rapid and selective detection of fatty acylated proteins using omega-alkynyl-fatty acids and click chemistry” J. Lipid Res. 51:1566-80.
Adapted from Yap et al. above...
Cells ( 1 °— 10 6 to 1 °— 10 7 ) were washed with cold PBS, harvested, and lysed with cold EDTA-free RIPA buffer by rocking for 15 min at 4°C. Cell lysates were centrifuged at 16,000 g for 10 min at 4°C and the postnuclear supernatants were collected.
[0.1% SDS, 50 mM HEPES, pH 7.4, 150 mM NaCl, 1% Igepal CA-630, 0.5% sodium-
deoxycholate, 2 mM MgCl 2 , EDTA-free complete protease inhibitor]
GFP fusion proteins were immunoprecipitated from approximately 1 mg of protein lysates (depending on the expression level of the GFP fusion larger amount of lysate may be needed) with affinity purifi ed goat anti-GFP cross-linked to Sepharose beads by rocking for 2 h or overnight at 4°C. ...the rabbit anti-GFP-sepharose sold by Abcam can also be used.
The beads were extensively washed with 0.1% SDS-RIPA, resuspended
in 50 mM HEPES, pH 7.4, with 1% SDS containing buffer or any standard SDS-PAGE loading buffer depepnding on the application or purpuse of the experiment, and heated for 15 min at 80°C (or 5 min at 95C or 2min at 100C (again depending on the application).
. The supernatants containing the fusion proteins were collected.
Please note that this would be a guideline only and may require some individual optiization.
Sam WasherAbcam Scientific Support
Answered on Sep 02 2013