Hi Andie. You can use the NC membrane for the detection of the Ki-67. If you specifically use the PDVF, You need to use the specific micron one to detect the high molecular weight. Try using NCM and use 300amp for around 6 hours as transfer conditions at 4 degrees.

HMW proteins are a pain no matter what! Both NC and PDVF should work. But far more important than the membrane you choose, is the composition of the gel and the transfer method. If these are not optimized your HMW protein may be stuck. See this article: https://tinyurl.com/2p8kh3xf

Hello Andie Woodcox

I would like to suggest the following.

For large molecular weight proteins, I would recommend using PVDF. Because large proteins can precipitate out in the presence of methanol, and PVDF membrane do not require any methanol in the transfer buffer, except prewet the PVDF membrane in 100% methanol for 30-40 secs before transfer. So, you have a higher chance of successfully transferring your protein to the blot using PVDF membrane. For larger proteins use PVDF membrane of 0.45um pore size as larger proteins can navigate through large pores more easily than smaller pores.

Semi-dry transfer systems have been considered ideal for mid-to-low molecular weight protein transfer, but suboptimal for high molecular weight protein transfer. Though the semi-dry technique is faster and consumes far less buffer, and being a tidier procedure, I would prefer the wet (tank) method for better transfer from gel to blot because wet transfer is less prone to failure due to drying of the membrane and is especially recommended for large proteins. In wet transfer, the sandwich is placed between positive and negative electrodes and submerged in transfer buffer to which an electrical field is applied.

Larger proteins transfer more slowly from gel to membrane, so you could extend the transfer time depending on the size of your protein of interest. The composition of the transfer buffer is also important. Since large proteins can precipitate out in the presence of methanol you should either avoid adding methanol to transfer buffer if you are using PVDF membrane or you could lower the methanol concentration to 5-10% in your transfer buffer. The presence of methanol does help as it promotes dissociation of SDS from the protein and dramatically improves adsorption of proteins onto membrane in the presence of SDS, besides helping to prevent gel swelling. You should also consider adding SDS to a final concentration of 0.1% because SDS adds uniform negative charge to proteins, making it easier for them to transfer from the gel and onto the membrane.

Because large proteins will transfer out of the gel very slowly, you can consider transferring for 90 minutes at 350-400 mA or overnight at 4°C at 40 mA.

I hope this helps.

Good Luck.

Thank you all for the answers so far! We have been using a 7.5% acrylamide gel from Bio-Rad instead of the regular ones. I've used only 10% methanol in the transfer buffer, have ran the gel for a long time so the protein can move down, and have transferred in the wet-tank overnight at 4C at 30V (these were done with nitrocellulose membranes). We are still getting lack-luster results. My PI wants me to try to use the semi-dry as it is faster and the procedure won't take as long.