Hello
I am working on a protocol to perform Western Blot on sorted endothelial cells from the liver. So far, I tried to do protein isolation in NP-40 buffer with 120.000 cells but I do not get any bands on the Western Blot. Does someone have a solution to perform WB on low cell count (100.000-150.000 murine sorted cells)?
Thanks in advance!
Hello Maxime Roes
You should try the following:
1. Be accurate in counting the cells.
2. Carefully handle the cell pellet.
3. Reduce the number of transfers of cells between tubes to minimize cell loss.
4. Centrifuging the cells in a swinging bucket rotor also decreases the risk of cell loss.
5. As mentioned by Andrew Lewis
use small volume of lysis buffer.6. Reducing the width of the well by trimming the comb teeth will improve the sensitivity of detection.
7. Use good quality antibodies.
Best.
Have you tried a Coomassie staining on your gel or Ponceau staining on your membrane to see if you actually have proteins? This will be important to know if there is another technical problem.
Did you measure your protein concentration? Don't load lower than 10µg/well.
Louis-Charles Béland
Dear Louis-Charles
Tried Ponceau staining on my gel, but did not see any clear bands. I triend to measure the protein concentration but the NP-40 is interfering with Bradford and is giving a wrong protein concentration for my sample.
Maxime Roes ,
You could try dosing your proteins by BCA, which is compatible with NP-40.
You could also try to stain your gel with Coomassie. The gel is not salvagable after the staining though, but will allow you to see if you had protein at that step of the WB.
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