I have performed transfection of n and p genes of ppr virus in vero cells.. Later i went for sds page followed ny westwrn blotring... After staining my page gel i could still see some bands on my gel.. Why they did not get transferred to my blot paper
Bigger proteins transfer more slowly than small ones. Also the higher % gel, the slower the transfer. In "normal" transfer conditions there will be always some proteins left on the gel. Increase transfer time (and possibly voltage) if you need more proteins on membrane.
Check with Ponceau S staining of the membrane that your transfer has worked.
It is nothing unusual, that you still have some protein in your gel left. The dynamics of the protein transfer are quite slow. To be safe you should of course check all your solutions an equipment, if there is some kind of issue). If there is no problem with your solutions, than you should just try to blot longer.
Bigger proteins transfer more slowly than small ones. Also the higher % gel, the slower the transfer. In "normal" transfer conditions there will be always some proteins left on the gel. Increase transfer time (and possibly voltage) if you need more proteins on membrane.
Check with Ponceau S staining of the membrane that your transfer has worked.
Couple of factors may cause your bands sticking to the gel instead of transferring. You may do the following-
1. Remove any bubble by pressing the gel-membrane sandwich properly.
2. Increase the time of transfer of protein from gel to membrane. Typically, 100V and for 1-1.5 hours. Due to excessive heat generated during transfer, make sure that while transfer takes place you keep your transfer buffer cool by adding ice pack and change the ice pack into a newer one after every 30 min. Best wishes.
It mainly depends upon the time of transfer. So even after transfer is done some of the higher molecular weight bands will be seen both on the membrane as well as in the gel.
The matter of concern is when the band corresponding to your protein of interest is seen in the gel but not on the membrane. In that case either the transfer time should be increased a bit or else the power pack and transfer buffer should be checked for any issues.
HMW proteins transfer better at lower methanol concentration in the transfer buffer and LMW proteins better at higher methanol concentration. Keep transfer at 0-4C and check transfer to the membrane with Ponceau.
For HMW proteins you can transfer at 30V overnight in a cold room (0-4C). The following day you should raise the voltage at 100V for 30 min.
Then stain the gel (with Comassie) to check if the transfer is properly occurred.
There is nothing unusual about it, but if you have problem in blots, may be not enough protein is being transferred. I prefer overnight transfer at 4 degrees celcius and 20-22V for assurance.
Blot membranes bind a certain quantity of proteins for mm2. If the gel is 1.5 mm thick it is difficult to transfer all. Anyway proteins move from gel to membrane because they are sds denatured , then 1) don't wash the gel in transfer buffer; 2) Don't let to dry it, 3) if you need to improve the transfer of high mass proteins add 1 ml of sds running buffer on the top of minigel-membrane following the direction of the current 3) check the watt during the blot because it should be >10. Be careful in using methanol because it is a fixative necessary to fix the protein to the membrane but can also fix the proteins to the gel. Viral proteins can be difficult to denature , we use a loading buffer with 3% SDS and 5% b-ME at final concentration. Some people use to add 6M urea in SDS_Page. Good luck
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