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Purified anti-GFP Epitope Tag Antibody
902601
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The aim of this experiment was to study the phosphorylation of Rb-analog, Whi5 in budding yeast after release from G1 arrest caused by pheromones. Whi5 was expressed as a C-terminal GFP tagged protein and its phosphorylation was detected by electrophoretic mobility shift in SDS-PAGE.
Western Blot
Cell Lysates from budding yeast
1:200 Dilution in 5% Milk (TTBS), overnight incubation in cold room
5% non-fat milk in TBS-T
Anti-Mouse-HRP, 1:3000, 1hour incubation at Room Temperature
None
Bio-Rad Clarity HRP substrate
Protein extract from cells was prepared in TCA lysis buffer supplied with PMSF, protease inhibitor mixture, and sodium orthovanadate. Lysates were quantified using a BCA assay kit (Thermos). 20 micrograms of proteins were subjected to SDS-PAGE and transferred onto PVDF membranes and blocked with 5% non-fat milk in TBS-T (20 mMTris, 500 mMNaCl, and 0.1% Tween 20) at room temperature for 1 h with rocking. The membranes were probed with primary Antibody (1/200 dilution) overnight at 4°C. After washing with TBS-T, the membranes were incubated with HRP-conjugated secondary Antibodies in 5% non-fat milk/TBS-T at room temperature for 1 h. The protein–Antibody complex was detected by Bio-Rad Clarity ECL substrate. The antibody detected endogenously GFP-tagged Whi5 as higher exposures (5-10min).
Works much better with overexpressed proteins
One of the very few good anti-GFP antibodies around
Not very strong with endogenously tagged proteins
I would use this for GFP detection in a variety of cell types for Western blot