I have been doing western blots on human brain samples since last 3 to 4 months regularly and the bands were coming pretty good. But all of a sudden I cannot detect any bands, even for Actin.I have tried every possible things and cross checked each step twice ( I tried changing the concentration of the primary, preparing mew primary, using new secondary, running new blots, using fresh buffers, changing washing time etc.) My Ponceau looks good. Please suggest some alternatives.
Thank You!
What detection method are you using? Presumably horseradish per-oxidase and ECL with a scanner or with film.
If you can't detect any bands, it may be a problem with the ECL substrate or the scanner or film.
@Brian Bower I have tried both; also I have used Li-cor without Che-mi ; but no bands detected. Although some of the sample (5 out of 11) do give bands with appropriate expression level but still not sure whats wrong.
@Thank You Ali
If 5 out of 11 samples show bands, I really do not understand what your problem is: clearly, all the reagents are working just fine!!!
Maybe the negative samples have undergone degradation, or maybe your target protein isnt present in those samples.
G
a few seconds ago
Utsav Nigam
Added an answer
Amar if actin is not coming, then the problem must be with either reagent or handling what I can possibly think of is , it must be primary or secondary so try to arrange a fresh stock or atleast ask for some of it from another lab and give it another shot. Good luck.
Amar Kumar
Did you fixed your problem because I'm also facing the same problem!! I have been doing western blots successfully for many months but in my last couple of blots, I couldn't detect any signal for my target protein but when I checked the loading control, the bands were good!!
Things I tried:
I prepared new dilution for my primary antibody still i couldn't detect any signal.
Tested my ECL substrate with my secondary antibody
Either your antibody has gone off
Your ECL has gone off
Or your lysates need to be re made (with freeze thawing they do not last indefinitely)
Laurence Stuart Dawkins-Hall Thank you for your answer but I'll clearly explain my problem.
Primary Antibody: I actually make small aliquots(0.2 ml tubes) for the primary antibody in order to avoid the freeze-thaw cycles. Now I made new antibody dilutions from both the primary antibody aliquots tube and also with the stock tube. Both showing no signal. My antibody is quite new and we bought it just 4 months back. I don't think it is the problem of primary antibody.
ECL substate: We use immobilon forte, I even tried with new substrate but It still there is no signal.
Lysates: I'm using new lysates only.
We use 1x TBST for blocking, primary(1:500) and secondary antibody(1:10,000) dilution.
Today I saw some white things floating in the 1x TBST bottle and so I'm thinking this could be the problem. I have to repeat all the things with these newly prepared 1x TBST but still This 1x TBST (prepared in 5 litres) is common for all the people in my lab and it works for others and also for me the loading control seems to be good!!!
Hi Suresh
Ppt in your TBS T is unlikely to cause this problem. Indeed it might result in non specific signal due to insufficient wash
A lack of signal as you ssy is not due to defunct reagents. That just leaves your lysate. If it is the lysate it is unlikely you would get a low copy number signal but might still het something from actin
How old is your lysate? Was it stored at -20c or -80c? How many times freeze thawed ?
If stored at -20c for more than 6 months; is lysate freeze thawed more than 3x especially from -80c. If low signal from say actin and no signal from lower copy number protein of interest then make new lysates
My lysates are new and I made it last week and the oldest lysates that I have is just 3 months old. I tried both the samples (Old and New). I usually store the samples in -20c.
I use lamin b as loading control
Laurence Stuart Dawkins-Hall Sir please follow this protocol i am using same protocol in my laboratory and it showed good band in brain protein this is abcam protocol so please find the attachment.
Hi Amit
This is an excellent protocol
Given that it has worked very well before this should not make a difference but in the absence of any further suggestions might be worth trying
In essence it is only necessary to coomassie stain your gel once with fresh protein lysates or with older lysates if you are experiencing problems. Sometimes the denaturation with acetic acid methanol coomassie blue can impair transfer and/or detection of your antigen. Thus
1. Soak your gel in tbst buffer for about 1 min and your pvdf membrane in methanol sution as you describe
2. Perform transfer for 2 hours
3. Verify transfer with ponceau S
4. When you come to ECL detect your antigen incubate with A and B solutions for 5 min
5. The only ither suggestion i can make is if this is a recent problem check your reagents for making lysates and verify protein cincentration by nano drop or bradford assay or qubit
Other than that not else what sure to say
You SOP is excellent and your reagents including antibodies are fresh
I notice you mention abcam who make about the best ab but also offfer ex ellent technical support
Certainely about 6 years ago when i was having problems i e mailed them and spoke to their tech support people by phone and they were very helpful
Laurence Stuart Dawkins-Hall
Hello Sir
I fixed my problem, When I reduced the volume of the protein sample, It actually works! I think it is because of the more material which takes up the substrate quickly and shows nothing in the blot! I understood that increasing protein amount does not necessarily provide us the solution to our problem. I hope it will be useful for others too!!
Good stuff
Generally you should use between 1ug and 30ug of protein lysate and no more
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