I cannot detect the western blot band of VDAC1, a Mito marker, in the whole naive T cell , but postive sample (VDAC1 protein) band is very clear.
I use RIPA to lyse naive T cell (about 6 M cell), centrifuge down, discard the pellet. The supernate add WB loading buffer to 100 Celsius degree 10min.
I used that sample to run SDS-PAGE and WB, but no band in this Lane.
I will very thankful if anybody tell me what the problem in the procedure.
Hey Qinjian Li,
your experimental setup sounds very good and robust to me. Especially adding a positive control is essential (so your antibody works, if VDAC1 is in your lysate). Also RIPA buffer is a good choice, because you want to detect a membrane-bound protein. So far this sounds exactly like our protocol for making Western Blot lysates with only one exception.
After heating the samples, we let them rest for a minute and the spin down very briefly and not long, because RIPA is normally so strong that it dissolves every membrane in your cell. You centrifuge for a longer time after heating when you want to get rid of nuclei and other organells when using a milder lysis buffer than RIPA. So there is no necessity for spinning down and removing the pellet. Perhaps you lose some proteins (including VDAC1) during this step.
I would give it a try to just spin them down very briefly after heating and make your SDS-PAGE and Western BLot just like you did before but with the whole lyste. Perhaps that already solves the problem.
I also checked some literature and (independent of the cell type) people load 40 up to 80 µg per lane when they stained for VDAC1. Perhaps you also need more material (meaning more cells).
I hope that helped you. Perhaps some VDAC1 specialists can add more info.
All the best,
Marc
Sometimes the protein is lowly expressed in the cells. Try loading higher amounts of the protein. It is preferable to load different amounts in increasing folds e.g. 25, 50, 100 ug and see the linearity of the band intensity. From these bands you can guess which lysate concentration to use for your experiment. Good luck
Are you get a band in THE SDS_PAGE gel or not??
And if you have a band in the gel, it may not be transmitted to the WB membrane.. In order to be sure, you can stain the WB membrane to see if the protein bands has transmitted into the membrane or not..
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