The problem that I'm having is not getting a heavy Ponceau stain OR detecting bands after I complete Western blot (addition of primary, secondary and chemi-luminescence) after transferring onto a nitrocellulose membrane. I've tried a couple of different things for troubleshooting: I've done a coommassie blue stain on the gel to make sure there is protein (despite the fact that I'm loading 40ug of protein)- and there was protein, I've doubled the protein concentration (to 80 ug of protein), I've tried varying thicknesses of the filter paper, and different transfer times (100V 1hr, 50V 1.5hrs, and 12V overnight). I'm unsure of where to go from here. Does anyone have any suggestions or ideas on what might be going wrong here?
What are the components of your transfer buffer? Are you adding enough methanol or too much?
It sounds like you are not getting transfer from the gel to the filter. Check that the voltage and current are what they should be. Make sure the orientation of the sandwich is correct, that you are not running the protein out the back of the gel instead of onto the filter. Stain the gel after transfer to see if the protein is still in it. Make fresh transfer buffer.
Hello,
The quality of the transfer depends on many factors; a) Trasnfer buffer --> I generally use Towbin buffer b) As Adam suggested, you should defintely check if the current and voltage are ok, generally for each half squaremeter of the gel, you can use 1-1.5 mA (Semidry Western Blotting). c) Do you use Tankblotting or Semidry (in that case the current values which I gave are different). d) Quality of your antibody --> Epitopes tend to degrade. You can check it for primary and secondary antibody e) Is your protein Tagged? Then, you can try to prepare two blots, one with an antibody directed towards the Tag and one with a specific antibody for your protein.
I hope these suggestion will help you to find a solution for your problem.
The above given suggestions would be helpful. Besides, you may try to load prestained protein ladder to make sure about the SDS-PAGE run and then transfer onto the membrane. In this way, you do not even have to go for ponceau staining as you can see stained bands on membrane.
Brandon: My transfer buffer has 16% methanol. Would that be considered too much?
Adam: My sandwich is in the correct orientation (membrane on positive terminal end and gel on negative end). I've tried staining the gel before and after transfer... before I have protein and after I don't so it's moving off my gel but little to none is actually sticking to the membrane. In terms of the voltage I always have it at a set value, but the amps fluctuate and tend to be low. Could this pose a problem even with a set voltage?
Can: Thank you for adding to what Adam said... I never knew about the mA/squaremeter I will check this to make sure that this isn't a factor that altering my transfer. I'm using a tankblotting method.
Pragna: My SDS-Page is running correctly... the protein ladder runs and transfers in addition to the fact that after I run the SDS-Page I stain with coommassie blue and there are protein bands on the gel everywhere.
By the way... thank you all for helping me out! I appreciate the feedback seeing that I'm new to this western blot technique. =)
your transfer buffer percentage is fine. Is your nitrocellulose really old?
All the suggestions so far should help, personally I always found semi-dry far more efficient and much faster.
The methanol concentration used can be changed depending on your protein size:
If it is small then 16% should be OK but you could decrease the SDS concentration slightly (small proteins don't bind to NC too well in th epresence of SDS). If it is very large then try reducing the methanol % (immobilizes large proteins in your gel) and increasing the SDS% (improves mobility of larger proteins) slightly.
Is your marker of similar MW to your protein of interest transferring OK?
Switching to PVDF should improve things as well as PVDF generally has a much higher binding capacity/cm2 and makes it more difficult to transfer the proteins right through the membrane and out the other side. There are also many different grades of NC with different surface charges, some much better for DNA than proteins so check which one you are using. If you want to stay with NC try adding a second membrane behind the first to capture anything that may have passed through-blot both if you have sufficient antibody.
If you are really laoding 10s of µg of protein it is far too much, your ECL(?) Western should detect 10s of ng levels of protein and overloading may even lead to a local 'white-out' where your protein of interest is (your film would look more like a negative of what you want).
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