I wanted to check the expression of a protein of 14Kda MW by Western Blot technique. I used 15% and 12.5% SDS PAGE gels and semi dry transfer method. No bands were visible even after loading 100 microgram of protein. Any suggestions on what should be the percentage of gel and what should be the transfer conditions. I have cross checked the antibodies and they are fine.
Dear Saha,
much depends on protein expression level, but in my experience both gel concentration are fine, simply make short runs to avoid proteins to exit gel (use colored ladders as reference). Also perform short transfers as small proteins may pass over the membrae (I do wet transfer). Finally reveal a positive control of similar MW e.g. Ubiquitin or SUMO
Hope will work
I agree with Alberto, the most important thing is to use a positive control in your case, because western can go wrong at multiple steps. (Maybe a histone if you can get some extract). If not, make sure you see other proteins in your extract like beta actin. I have used a 15% gel for a similar purpose so that shouldn't be a problem.
Another important thing to consider is your detection technique. Firstly, how have you crossed checked your antibody? Because if you have validated by immunofluorescence or IHC it still may not work well for western. Secondly, have you made sure your secondary are fine (put 1ul in ECL mix and check for fluorescence)? A beta actin positive control would tell you that your transfer and your technique is fine but your protein in undetectable due to antibody. Thirdly, considering your antibody is fine, try detecting your protein in gel by silver staining to make sure it is present. I have detected histone H4 by this method before proceeding towards blotting and found it to be a very important quality control. With the amount of protein you are loading I dont think this should be a problem (This however depends upon the way you have extracted your protein. If its a total cell lysate, you still should be able to see bands of proteins with similar sizes). Also make sure your washes are not too harsh and your blocking is compatible.
Transfer: I have used we transfer by biorad for a 15% gel to detect a 20KDa protein. (100mV-1.0 hrs), blocking- 5% milk in TBST, washed with TBST for 10 minsx3 after each antibody.
I hope this helps :)
I'm not sure about semi-dry blots, but I have gotten this to work with fully "wet" tank transfer protocols before.
Used a high conc. of methanol in my transfer (20%). Also short transfer times, an hour at most though sometimes go with 30 minutes. And lastly, try adjusting the voltage a bit as well. High voltages can cause some low MW proteins to be forced through the membrane.
Lastly it is not unheard of to use multiple membranes in a western. This increases the likelihood that at least one of the membranes will catch a low MW protein.
Best of luck
Thanks for your suggestions Rachayata.
As you mentioned rightly, I have used Beta actin as control and have got the desired bands. Although I didn't do silver staining of the gel separately, I have stained the gel after transfer in coomasie staining solution. The lower molecular weight proteins were not present in the gel after transfer, so that suggests transfer isn't a problem. It might be that protein has passed through membrane to filter paper. I kept for 25 mins at 135mA (12.5%) and also 30 mins 120mA (15% gel). But we do gamma H2A.X blots in similar way and do not face a problem. Also, I used minimal wash for it. For blocking I used 5% skim milk in TBST. I used a higher concentration of antibody as well.
Try to reduce and denaturate your sample and spot it directly on membrane. Also use an highly expressing sample (eg cells expressing your target proteins or recombinant protein). If this doesn't work there are not many possibilities
1) the ab you are using is not suitable for WB
2) protein level is too low.
Thanks for the valuable suggestions Alberto and Thamsanqa. But it is not a recombinant protein. We needed to check the difference in level of expression after drug treatment. Naturally it is a more or less abundant protein and I feel expression is not a problem. I agree if protein estimation is incorrect, may be amount on the gel is too low to be sensitive for the antibody interaction. We do have a positive control though, but even for that, I'm not getting the expected band.
What do you mean: I'm not getting the expected band?
No band at all or an incorrect band size?
Do you know if your protein may be modified? E.g. multiple phosphorilation, sumoylation ecc...
All low proteins are absent after transfer? Did your molecular weight standards ran and transfer OK?. First you have to find the bands before thinking about the antibody.
You have to verify if your Ab is suitable for WB, load a known high expressing sample. Whatever it is, cells, tissue or a positive control bought from a manufacturer.
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