I have been working with GFP that I have exprrssed in HEK and HeLa cell lines. On transfer I can only see the marker on the nitrocellulose. I cannot even see the positive control on the nitrocellulose. I made a fresh batch of antibodies and also increased its concentration but nothing has worked so far. I mean ECL provided no results. Anyone has any explanation for this?
Hi Vishwesh it sounds like you are loading too low an amount of protein (as the marker is transferring it does not sound like a transfer problem).
Have you probed with your ab for loading control (I,e, actin/GAPDH)?
Also do you check for protein content in your lysate before loading (such as Bradford assay) or after transfer on the blot itself (i.e. Coomasie)?
Cell signalling have drawn up a trouble-shooting guide which might help
https://www.jove.com/video/5071/western-blotting-troubleshooting-guide-cell-signaling-technology
Hope this helps,
Gary
I did check GFP presence using agarose gel (using restriction enzymes). I did observe the expected bands. I then transfected the mammalian cells with pDNA. I have used a rabbit GFP(antibody) and a secondary IGG. Except the markers, rest fails to transfer.....or the ab doesn't work. What would be your guess?
the amount of marker loaded is actually less than the control and the samples.
marker- 5ul
+ve control- 10ul
samples- 15ul
Hi Vishwesh - the GFP presence you are checking is the DNA presence.
I was referring to the protein you are loading.
Did you check for protein content before loading the lysate into the lanes?
If so how much protein did you load? Did you check for loading control with i.e. an Actin antibody? As mentioned I suspect that you are not loading anything but lysis buffer.... but you will not know unless you check. Performing a Coomasie stain on the failed membrane will also tell you if any protein has transferred (or not).
I performed the coomasie stain. the proteins have failed to transfer. thank you for your help!
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