I am trying to do a western blot and the protein transfer is confirmed by Ponceau stain. However, I never get bands on the x-ray film. I am new to the lab, so I started with b-actin standardization before I actually do my experiments.
Here is the protocol used:-
Transfer:-
Day 2:
Other details:
1. Cells used for cell lysate preparation- human peripheral blood mononuclear cells PBMC. (lanes 2-7) plasma samples lanes (8-9) lane 1 and lane 10 ladder
2. Blocking- 5% non fat dry milk in TBST
3. Primary Antibody- beta-Actin Antibody Catalog (MA1-140) FisherScientific.
4. Secondary Antibody- Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP Catalog #62-6520 FisherScientific
5.Chemiluminescence substrate- EMD Millipore LuminataTM Crescendo Western HRP substrate
6. Precast gel 7.5% from Biorad was used.
Results documented:
Figure: Ponceau stain of the blot to confirm that transfer of proteins occured
X-ray sheets:
No bands were developed.
Entire x-ray sheet turned black :(
Hi,
If the entire x-ray sheet turns black as you write (not only the part exposed to the membrane during development), could it be that the film was exposed to light already prior to development?
If you are sure this is not the case, or if it is just the area of the film covering the membrane, it might be a problem of blocking/washing or antibody dilutions, though your protocol overall seems fine.
I suggest you try:
A new set of films (or one you are sure was handled appropriately, i.e., in the dark), or another development method. Do you have access to a chemiluminescence detector (some gel documentation systems can do this too)? Alternatively you could try a colorimetric method with DAB substrate. Maybe not as sensitive or pretty as X ray, but it should do the job as well for b-actin, and could confirm all steps before the development are ok.
Hope this helps,
Carla
Hi Carla,
Thank you for the prompt response.
I don't have access to chemiluminescence instrument.
The pack of film was new and opened only in the dark room.
I will try next try different dilutions of antibodies and maybe use BSA as blocking solution instead of milk.
Thanks,
Anshul
Looks like your protein quality is not so good. Bands after Ponceau look smeared. Do you have protease inhibitors in your lysis step? Your overall protocol looks fine.
Also if your entire blot is turning black, you might want to block for one hour and then reduce your primary and secondary antibody concentrations. Of course, you should check the film first.
Hi Rekha,
Thank you for the inputs.
I use commercial RIPA buffer for lysis. I will check if it has inhibitors. And will add inhibitor cocktail if it does't have inhibitors.
I block for one hour and also tried blocking overnight at 4C.
Reducing the primary and secondary antibody concentrations will be my next step.
Thanks and Regards,
Anshul
Hi Anshul,
The protocol looks fine.However, I do not see any protein except in the last two lanes (which looks degraded too).
The problem seems to be in the sample. I would recommend to use a positive control apart from your samples. You can also stain one gel with Coomassie to check your samples first before proceeding with western blot.
Also, check your developer and fixer if your X-ray film is turning black.
Good Luck!!
Ruchi
Thank you Ruchi!
The picture of Ponceau was taken from phone and not a good one. There were varied concentration of bands in all the lanes, but relatively less compared to the last two lanes.
I am going to run a new set of samples. and try making the suggested changes.
Thanks
Anshul
Hi Anshul,
I hope you are doing well? Coming to your blot...protocol is ok. Please review these things:
1) Check the usages manual of antibody and its working condition.
2) Block your membrane 1hr using 5% skimmed milk at 37o C.
3) in the last two lane there seems to be overloading of protein so reduce.
4) Control the exposure of the x-ray film and also the developing and fixing buffer, they go bad with time or exposed to light.
Since you are using colored marker I think you can bypass ponceau stage, sometime ponceau inhibits antigen antibody interaction.
You can text email me or text me your number...I can discuss the problem with you.
Nice to see you after long time and good luck.
There are a couple of things I'd do differently:
- Make sure you have appropriate protease inhibitors in your lysis buffer
- Do not boil the sample, but heat to 60 °C for 15'.
- Use PVDF instead of NC, it has greater protein binding capacity and -affinity, and it doesn't fragment during incubations. Don't forget to pre-soak the PVDF for a few seconds with methanol, then dd water and finally buffer.
- Use Dunn's transfer buffer instead of Towbin's (doi:10.1016/0003-2697(86)90207-1, 10 mM NaHCO3, 3 mM Na2CO3). It not only works better, but is cheaper too. For large transmembrane proteins add 50 µM SDS.
- At least in my hands, tank blotters work better than semidry, probably because the gel isn't heated so much.
- Complex mixtures of proteins, like non-fat milk powder or fish skin gelatin, work better as blocking agent than single proteins like albumin. Recall though that milk proteins are phosphorylated and can't be used with anti-phosphor antibodies.
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