Thermo Fisher Scientific
V5 Epitope Tag Antibody (E10/V4RR)
MA5-15253
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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 endogenously C-terminal 3xV5 tagged protein and its phosphorylation was detected by electrophoretic mobility shift in SDS-PAGE.
Western Blot
Cell lysates from budding yeast
1:5000 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
Biorad 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 (1/5000 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 endogenous levels of Whi5-3xV5 protein quite robustly.
Great antibody for V5 epitope
I would use it every time for detection of V5 epitope tag.