Hi
I lysed rat hippocampi neurons and ran a western blot for B-III Tubulin protein and I saw protein bands. However, I reprobed the PVDF membrane (after three washes with TBST) with my housekeeping protein GAPDH and saw no signal. I reprobed again the next day with a higher concentration of the GAPDH antibody but I still fail to get any signal. Does anyone know why that is happening?
Hi, you ran SDS-PAGE and then transferred to PVDF membrane, stained with primary anti- B-III Tubulin and secondary antibody, and then you saw protein band. If this is the case, you have to use striping buffer (can follow the protocol from the following link or from one of my previous publications) to reprobe
Article Identification and Characterization of Novel Perivascular Ad...
Somasundaram C et al. 2012
https://www.google.com/search?q=strip+buffer&oq=strip+buffer&aqs=chrome..69i57j0l5.4728j0j7&sourceid=chrome&ie=UTF-8
Stripping Buffer: To prepare 100 ml, mix 6.25 ml of 1M Tris-HCl pH 6.8, 10 ml of 20% SDS and 700 μl β-mercaptoethanol. Bring to 100 ml with deionized H20.
You have to use it under laminar flow hood
Were both antibodies raised in same species? You haven't mentioned it. If you are using HRP conjugated secondary antibody then HRP inactivation protocol you can follow.
Once probing the target antibody, wash the blot 3 time 10min each with TBST, incubate the blot in 1mM sodium azide solution in 5% skimmed milk at 4 degree overnight. Next day again wash 3 times 10 mins each with TBST and go on with next antibody raised in different species.
Hope this might help.
1. stock anti-GAPDH went wrong.
2. your sec for it is inappropiate/went wrong.
i guess you see the signal of first labelling. if not, even the detection reagent went wrong somehow.
try to probe against actin, and check the secondary you need.
I have previously done a western blotting for GAPDH so I know the primary and the secondary antibody works.
For this experiment, I ran a western blot for b-III Tubulin, that is transferred from gel to PVDF Immobilon and stained with primary and secondary antibodies. The antibody for B-III tubulin is raised in mouse and secondary is Alexa Fluor 488 Anti-mouse.
After seeing bands for B-III Tubulin, I simply washed with TBST and stained with GAPDH which is also raised in mouse.
Thanks a lot for your comments! After reading all your answers, I stripped the same membrane (PVDF Immobilon) with 0.5 M NaOH and wash it once with TBST and then with TBS.
Following this I blocked with skimmed milk in TBST and stained with anti- mouse GAPDH. I still did not see any bands though.
Does anyone have more suggestions on why this hat this could be happening ?
Hi Sushmita
It might be that even after stripping, that membrane has way too many probings, if that makes sense. The GAPDH binding did not work the first time, hence now it is kind of tricky to re-probe it again. And every time you block you kind of create a canvas for that secondary antibody to find your GAPDH, but it is not there.
Also, in my opinion, stripping protocols are mainly useful to remove all the secondary signal of your membrane. It is normal to see faint bands of your first protein after stripping for your second protein. In my opinion I always prefer to cut the membrane after the transference, and blot each piece of my membrane with my proteins of interest. Also for papers and quantification purposes, stripping might be not desirable. Unfortunately sometimes this is not possible as the MW of your proteins could be very close. You can play with the gel concentrations to separate more your proteins but maybe that is a route you would not like to go to.
Another reason why possibly your stripping did not work is because you are using a low concentration of NaOH. I have seen even 2M of NaOH. Nonetheless, sometimes very harsh conditions migh result in completely loose of protein. In my case, i never used high basic solutions such as Naoh to strip my membranes, I always use mild acidic solutions as they are way more effective to kill the signals form antibodies and reprobe your membrane again. I will copy here my stripping protocol with which i always had very nice results (it is available in abcam's website):
buffer preparation:
7.5 gr glycine dissolved in 200 ml of Milliq water
adjust PH to 2.2 with concentrated HCl
add 0.5 gr of SDS
add 295 ml of milliq water
stirr and dissolve
add 5 ml of tween 20 and mix gently
stripping protocol:
-immerse your membrane in stripping buffer, shake vigorously for 10min
-discard the buffer and repeat other 10min
-discard the buffer and wash membrane for 10min with pbs1x (repeat this 2 times)
-discard the buffer and wash membrane for 5min with tbst (repeat this 2 times)
-block the membrane with milk
-incubate with your antibody
it might seem a lot of washes!! but after stripping you need to be sure your membrane is completely clean as if it was fresh, as your antibody has to bind to your new protein without interfering with whatever was there before. I add SDS and tween on my buffer for that reason, they both really help to wash and remove previous interactions. I would try with your old membrane, but if you could use a new one that would be the best to judge. Hope this helps!!!
I would caution against stripping. Try re-activating the membrane with methanol (~20min or so should be sufficient).
Rishi Agrawal, Do you submerge the membrane in 100% methanol for 20 minutes and rinse withTBS? After that, I guess you can develop again with ECL substrate right?
Thanks
Hi! First of all, you have to activate your PVDF membrane in metanol. Then, you can use it normally.
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