Hi everyone,
We are doing western blot with yeast protein extracts. We are applying a wet transfer method using Tris-Gly as a transfer buffer (with 10% methanol) and running it with constant current (400 mA) for 2 hours at 4°C, while the tank is put in a box with ice. To check the transfer Ponceau staining is performed on the nitrocellulose membrane and we checked the gel (12% SDS-PAGE) with Comassie Blue staining as well.
As you can see from the photos, there's a lot of protein that are still in the gel and it seems that we had an uneven transfer from the gel to the membrane (there are more HMW proteins than LMW proteins).
What could cause it? Maybe is it related with protein concentration? (there are 50 ug of proteins per lane). Is it possible to transfer all the proteins from the gel to the membrane?
The cell lysates were obtained with whole-cell method.
Thanks,
Michele
Hello Michele
Please consider the following points:
1. First and foremost, the amount of protein loaded per lane is too high. You should load about 15ug to 30ug protein per well. The gel can hold more of the protein than the membrane binding capacity. Therefore, the excess protein may stay in the gel.
2. Higher percentage gel can slow the transfer process. So you need to increase the transfer time and if required even the voltage. Since you have both HMW and LMW proteins you need to optimize the transfer conditions.
3.Methanol is a fixative generally used to improve the adhesion of smaller proteins to the membrane. Since you have more of HMW proteins you need to lower the percentage of methanol in the transfer buffer. Also the binding of protein to the gel will be reduced.
Good luck.
Hello Michele
Please consider the following points:
1. First and foremost, the amount of protein loaded per lane is too high. You should load about 15ug to 30ug protein per well. The gel can hold more of the protein than the membrane binding capacity. Therefore, the excess protein may stay in the gel.
2. Higher percentage gel can slow the transfer process. So you need to increase the transfer time and if required even the voltage. Since you have both HMW and LMW proteins you need to optimize the transfer conditions.
3.Methanol is a fixative generally used to improve the adhesion of smaller proteins to the membrane. Since you have more of HMW proteins you need to lower the percentage of methanol in the transfer buffer. Also the binding of protein to the gel will be reduced.
Good luck.
Michele Mario Gennari I agree with Malcolm Nobre
Only load more than 40 ug protein if the protein you are detecting has a low abundance.
For HMW proteins, you need to run your transfer for longer (O/N) at a low voltage and low temperature. Typically I run my transfers at 4 oC at 30 V for 720 minutes.
Consider adding 0.1 % SDS to your transfer buffer to reduce precipitation and reduce methanol to 5 %.
Thank you Malcolm and Jack for your answers! I'll definitely consider your suggestions to optimize my protocol.
Hi Michele
I will point out certain things here:
A. I noticed the bubble presence (in the first lane from left and less prominent in the third lane of ponceau S) in the membranes probably indicating that the sandwich of filter paper, gel and membranes might not have been made accurately.
B. 10% methanol is fine for transfer but i think 2 hrs transfer time is still low. Maybe you should try slow transfer and corresponding adjustments in the current and/or voltage of transfer.
Hope it helps!!
If you want to look at the high mw proteins, be aware that they migrate slowly...in the first dimension and when you transfer. This can be solved by using lower percentage gels or running those higher percent gels longer...to get the high mw proteins further into the lower percent range of a gradient gel. Transferring by tank, slow and cold (even overnight in a cold room) may provide consistency. Membranes vary by chemistry. Take a look at a good commercial trouble shooting guide associated with the set up you are using. Nitrocellulose may not be optimal if you are looking for hydrophobics or something that may co-migrate with many other proteins of the same size. Procedures that remove any "masking co-migration proteins" (like linking the transferred proteins to the membrane and pre-stripping "masking co-migration" from the transfer .... which can assist opening up the epitopes for the ab incubation... ) can be completed before blocking for the first probe. Be sure to wash thoroughly if a stripping buffer with high SDS, heat and a reducing agent are employed in the test for "availability" of the antibody binding sites. "More" protein in western blotting is often not good. Less is more if you have co-migration bands.
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