I want to detect BAD and pBAD (23 Kda size) and CREB and pCREB (43 Kda size) from brain samples of hamsters....When i do western blot with normal protocol giving 80 voltage for 4 hours to transfer protein from Gel to membrane i only detect Pan Calcineurin 59 KDA but none of other protein....is it because of longer time as i detected some of the mentioned protein when i gave 100 voltage for 2 hours? Does longer transfer times take protein out of membrane? is it just something related to antibodies?
Dear Zahid are you using Bio-Rad Semi-Dry transfer? if yes, please be careful that the voltage is constant 18V for transfer (shouldn't exceed). For PrP which is of 26kDa, I use 18V (constant), 0.06A (60mA) per 10-17 wells gel in semi-Dry transfer for 55-60min. If you are using 20-26 wells gels, then increase the current. It should work for even the proteins of your interest (I have used it for proteins up to 60kDa and it works well).
Good luck
Thanks...Salamat brother i dont use semi dry...i am using 12% gell and then wet transfer...
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