Hi,
USed ANy KD gel from Biorad. Gel run was good. Transfer was not efficient, bands were still appearing on the gel but with low color intensity. I used TGS to transfer (20% methanol), 60V for 90min.
Hi,
Pedro Paulo Gattai made some good points. Here are a few other things to think about or try:
-did you pre-wet the PDVF membrane in pure methanol and then equilibrate it in transfer buffer before use? All you pads and paper should be wet before use as well
-do you rinse the gel before making the sandwich to get rid of excess SDS if that is in your gel system
-when you are making your sandwich, do you press out all air bubbles using a clean pipette (or other device) to make sure there is good contact between all the layers?
-if you use a pre-stained marker on your gel, does most of it get transferred to your PVDF membrane?
-is your transfer done in the cold- in 4°C cold cabinet with the ice pack in the transfer container (if one is available)? pre-cool the transfer buffer as well
-you could try a much lower voltage (say 15 V or whatever is the lowest on your power-pack) and allow the transfer to proceed over night in the cold
-if you stain the gel after the transfer is complete, is there protein still in the gel-maybe the transfer is not efficient only above or below certain molecular weights-try a different cross-linking percentage; have you tried staining the membrane to see if the proteins are there?
-have you tried lowering the amount of methanol (or using ethanol) in your transfer buffer? the hydrophobicity of your protein of interest may not require it
-have you tried the complete Western (or whatever your detection system is) procedure-just because you can't see the protein with coloured stains doesn't mean it is not there-the antibodies will be much more sensitive
Good luck!
do you noted if the sequence of gel and membrane is it right? I mean, paper, gel, membrane, and paper inside the sandwich? Other thing that you must pay attention is if you put this sequence (paper, gel, membrane and paper) to transfer from negative to positive (from black part of sandwich to transparent one).
Did you saw if the paper after the gel has marker of your standard?
Other thing that can affect your result is about the voltage that you used, because we usually use a voltage of 50V not 60V.
I hope it helps you.
Hi,
Pedro Paulo Gattai made some good points. Here are a few other things to think about or try:
-did you pre-wet the PDVF membrane in pure methanol and then equilibrate it in transfer buffer before use? All you pads and paper should be wet before use as well
-do you rinse the gel before making the sandwich to get rid of excess SDS if that is in your gel system
-when you are making your sandwich, do you press out all air bubbles using a clean pipette (or other device) to make sure there is good contact between all the layers?
-if you use a pre-stained marker on your gel, does most of it get transferred to your PVDF membrane?
-is your transfer done in the cold- in 4°C cold cabinet with the ice pack in the transfer container (if one is available)? pre-cool the transfer buffer as well
-you could try a much lower voltage (say 15 V or whatever is the lowest on your power-pack) and allow the transfer to proceed over night in the cold
-if you stain the gel after the transfer is complete, is there protein still in the gel-maybe the transfer is not efficient only above or below certain molecular weights-try a different cross-linking percentage; have you tried staining the membrane to see if the proteins are there?
-have you tried lowering the amount of methanol (or using ethanol) in your transfer buffer? the hydrophobicity of your protein of interest may not require it
-have you tried the complete Western (or whatever your detection system is) procedure-just because you can't see the protein with coloured stains doesn't mean it is not there-the antibodies will be much more sensitive
Good luck!
Hi
Although Robert C Richards have mentioned all possible reasons. You can try nitrocellulose membrane for Western. You can also increase the transfer time and check the transfer by using pre-stained markers.
Thank you all or your valuable suggestions. I just finished my second trial, found that the transfer was good. But to my surprise, some bands from marker lane was still visible on the gel with reduced color intensity.
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