western blot 转膜时间与样品蛋白质分子量的关系

western blot 转膜时间与样品蛋白质分子量的关系 1. For transfer of proteins smaller than 20 kDa, transfer proteins from gel to PVDF (polyvinylidene difluo ride) membrane at 1Amp constant current for 45 mins or equivalent (250mAmp for 3 hours or 500mAm p for 90 minutes) in transfer buffer (25mM Tris, 190mM glycine, 20% MeOH). 2. For transfer of proteins smaller than 120 kDa, transfer proteins from gel to PVDF membrane at 1Amp constant current for 1 hour or equivalent (250mAmp for 4 hours or 500mAmp for 2 hours) in transfer b uffer (25mM Tris, 190mM glycine, 20% MeOH). 3. For proteins larger than 120kDa, transfer to PVDF membrane at 1Amp constant current for 90 minute s or equivalent (250mAmp for 6 hours or 500mAmp for 3 hours) in transfer buffer + SDS (25mM Tris, 1 90mM glycine, 20% MeOH, 0.05% SDS). 4. For Proteins larger than 250kDa, transfer to PVDF membrane at 1Amp constant current for 1 hour an d 45 minutes or equivalent (500mAmp for 3.5 hours) in transfer buffer + SDS (25mM Tris, 190mM glyci ne, 20% MeOH, 0.05% SDS). Add transfer (or CAPS) buffer to the transfer unit according to manufacturer1s directions. Transfer over night with a setting of 20V / 40 mA or for 3 hours at 70V/160 mA. The transfer process needs to take pl ace under cold temperatures to prevent the gel from sticking to the membrane. This can be accomplish ed either by using a water cooling core in the transfer unit or placing the entire unit at 4?. Please follow the manufacturer1s recommendations