As i am facing this problem, i changed the homogenate (kidney) and made new. For electroscopes , i used 10% SDS-PAGE and sample volume was 50 micogram/10 micro lit.. For transferring protein i set the volt at 100 and 60 min. I can see the standard protein band but not the desired band after staining with Ponceau S. Please kindly suggest me to solve this problem .
Dear Nasiruddin,
If you are unable to see the desired band on your membrane after Ponceau stain, it clearly means either your protein is not being transferred on the membrane or your lysate doesn't contain the protein at all (which is very unusual). So, assuming the problem lies with the transfer, I would like to know the molecular weight of your protein. Depending on which you should standardise your conditions of transfer. If your protein is too big then you should increase the voltage and time of transfer. If your protein is too small, then may be you are over doing the transfer and your protein is pushed across the membrane, in that case reduce the time and voltage and also may be use a higher percentage of SDS PAGE. Moreover, which transfer method (wet, semi dry) do you use? This would also affect your efficiency of transfer.
Dear Tias saha, my desired protein is about 68 kD and i am using the wet transfer method.
Agreed this sounds of a transfer issue. A useful control is to coomassie stain your gel after transfer to see what has been left behind. Also I've found using PDVF, although more expensive than nitrocellulose, is so much more reliable it outweighs concerns over the cost. With PDVF, I've found it difficult to over transfer (except for proteins around 15k or less) and I routinely use 0.5 Amps for 1 hour. Cooling the transfer is safer but I've found no evidence that heat damages the transfer when using PDVF.
thanks for your suggestion. But , i would like to know is there any suggestion regarding protein concentration or addition of SDS in transfer buffer ?
50 ug is about the largest I've ever heard loaded on a western. SDS definitely can make a huge improvement on transfers. We use 0.01% in our lab. We've tested and many proteins simply won't transfer without SDS. Other labs use significantly more or less. In our case we've used those coomassie stains as a standard for whether we are getting full transfer
Thank you for your suggestion. I have tried 30V overnight with out addition of SDS in transfer buffer, have seen only one band of low molecular weight protein (~20kD) by membrane staining . I will try by addition of SDS as you mentioned.
Try loading samples on two SDS gels and run them simultaneously. Stain one with Coomassie and leave the other for Western transfer. If it shows up on your Comassie stained gel, then you know its present. Next try out above suggestions, keep it cold while transfer. To optimize transfer, try with different size proteins while covering your size range to know what conditions works best. Once you have figured out, then just transfer the desired protein.
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