Cell Signaling Technologies
SirT1 (C14H4) Rabbit mAb
#2496
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Here, we detected expression levels of SIRT1 protein in primary human CD4+ T-Cells before and after Treg differentiation.
Western Blot
Primary Human CD4 cells
Dilution 1:2000 (1% Milk in TBST), incubation overnight, 4°C, wash
Block with 5% Milk in TBST for 2h
Dilution 1:2000 (mouse anti-rabbit), 1% Milk, 2h, room temperature, wash
Signal Fire ECL Substrate, 30s
Chemoluminescence, detected with X-Ray film
The above mentioned SIRT1 antibody works quite well with higher dilution as the manufacturer's recommendation. After using the ECL substrate, the X-Ray film was exposed to the membrane for 2 minutes and 30 seconds.No unspecific bands were detected using this antibody on CD4+ T-cells. The predicted band size was in line with the observed band size.After stripping, it was possible to reprobe the membrane if desired.
In our opinion, TBST (0.1% TWEEN-20) works better than PBST. Membrane equillibation in ECL substrate for 30 secons enhanced chemoluminescence.
Small amounts of antibody required
As always, antibody titration needs to be conducted.
Easy to use, quite reproducible results. Membrane reprobing is possible.