Sepharose Anti-GFP antibody

• IP

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Immunoprecipitation - Sepharose Anti-GFP antibody (AB69314)

Lane 1 & 3 : IP from COS 7 cells transfected with EGFP.N-Ras (48 kDa)

Lane 2 & 4 : IP from untransfected COS 7 cells

Lane 1 & 2 : IP using 15 ul of rabbit anti-GFP conjugated to sepharose beads (0.5 mg IgG per ml of beads)

Lane 3 & 4 : IP using 15 ul of goat anti-GFP conjugated to sepharose beads (1 mg IgG per ml of beads)

Immunoprecipitation : COS 7 cell lysates containing 100 ug of total protein in 200 ul of 0.1% SDS-RIPA buffer with addition of complete protease inhibitor were used for each immunoprecipitation. Cell lysates were incubated with anti-GFP sepharose beads for 2 hours at 4oC with rocking. Beads were washed. Proteins were eluted with 1% SDS 50 mM HEPES (pH 7.4) at 80oC (15 min). Half of the IP sample was loaded on each lane of 10 % SDS PAGE gel and gel was processed for Western blotting/ECL.

Western blot : Primary antibody : monoclonal mouse anti-GFP (ab291) at 0.2 ug/ml in 5% non-fat milk/TBS-T; 1 hour incubation at room

All lanes:

Immunoprecipitation - Sepharose Anti-GFP antibody (ab69314)

Predicted band size: 27 kDa

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