Hi I transferred my protiens from gel to the blot but the ladder was not found completely transferred can anybody suggest me the reason?or could it be because of voltage setting?
If the MW standards are not fully transferred, it is possible that the contact between gel and the membrane was not good enough. Perhaps you did not get rid of an air bubble on the upper part of your gel? Heavier proteins transfer worse than the light ones in general. Also, you could have loaded more of your protein samples relative to the MW. How big are your proteins? If they are smaller than the MW bad standards that did not transfer, then you are OK. If they are in the same size catgories, air bubbles or anything else that could disrupt the transfer might have happened. I suggest to repeat the experiment and see if it happens again.
As Scheherazade wrote...the high MW bands transfer more slowly...I would suggest that the transfer was not long enough or high enough Amp/volts, or was possibly interrupted. I transfer my WB for 2.5h at 250mA. If you are using PVDF membrane dont forget to soak in Methanol prior to setting up your transfer. Alternatively, as mentioned above, it could be a large bubble over the area of the ladder that prevented it transferring, however if care was taken to get rid of bubbles when setting up the transfer then this is less likely.
dear friend, what happens is normal, proteins move from the gel for their molecular mass and for their charge. If you want to increase the transfer of high molecular weight proteins, you have to do a less concentrate gel, or you have to add 1-2 ml of SDS running buffer (with sds) on the gel in the opposite side of the membrane. Methanol is used only to fix proteins to the membrane. In my opinion when you soak the gel in the transfer buffer containing 20% methanol you risk to fix the proteins to the acrylamide-matrix and that they do not move at all from the gel. Anyway all transfer manuals have this, incomprehensible to me, soak step.
It is perfectly normal that the proteins with higher MW take more time for migration from the gel to the membrane. As already mentioned, it depends on the size of your proteins of interest. If they are of similar size as the marker bands which were tranferred well, you can keep your settings. if they are in the bigger region, you have to increase transfer time. A longer transfer time increases the transfer efficiency for the bigger proteins, but you have the risk that very small proteins are blotted through the membrane and get lost, so you really have to decide which size range is most interesting to you. If you need investigate a relatively big size range, you can try a discontinous buffer system which should reduce the impact of protein size on transfer time.
Paola: The use of Methanol for PVDF membrane is unavoidable as it is very hydrophobic. If you use water/aequous buffers without previous methanol treatment PVDF does not expose its full protein binding capacity. I believe Methanol in transfer buffer helps bind small MW proteins to the membrane...
'Methanol in the transfer buffer aids in stripping the SDS from proteins in SDS-PAGE gels, increasing their ability to bind to support membranes.'
That means you haven't transfer the total protein onto the membrane. You'd better increase your transfer time.
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