I'm working on the expression of gH2AX protein in primary cells using the western blot. unfortunately, with normal ECL I couldn't detect any band! and with sensitive ECL I just see false positives. even between lanes!!! my method is:
gH2AX : 17 kDa
Gel%:4-12
Loading: 20ug protein
Transferring: 1h 25v
blocking buffer NFDM 5% (1.5h/RT)
Primary Ab: O/N 4C
• Rabbit monoclonal Anti gH2AX, cell-signaling/9718s 1:1000
• Secondary Ab: 1h/RT
Anti-Rabbit IgG, Cell signaling, 7074, 1:5000 1h/RT
does anyone have a similar experience?
Hello Maryam there could be many things to check like:
1. Anti gH2AX, cell-signalling/9718s= is a phospho form antibody it will not show you the normal gH2AX.
2. For 17 KDa protein, I will recommend you to run a 12-14% gel.
3. Please use 4-5% BSA for blocking (at least 1 hr).
4. After blocking please wash with TBST, pH 7.4.
5. Check your Transfer time, since it was a small protein, you should run for 90-95V (around 100 min)
if *1* is okey then kindly check point number*2* 3* 4*5, I am sure it will solve your problem
Do you get a signal in a dot blot or ELISA like setup? Do you have a positive control?
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