Proteins are also stripped a little every time you strip your blot so there is a limited number of times you can strip a blot. I have some antibodies that won't work on a stripped blot but work perfectly on an unstripped blot. I probe with these antibodies 1st and then strip and reprobe with the stronger antibodies like Tubulin, Actin, or viral proteins. Make sure you wash your membrane throroughly after stripping step and block again for minimum 30mins.

My transfer buffer recipe is:

6.04 g Tris and 28.2 g glycine and add to 2 L bottle. Add deionised water to 1600 ml, then add 400 ml methanol. The pH should be 8.3. (I never cheack the pH though as its always correct).

Try incubating your antibody on your membrane in a containing the size of your membrane and have about 10ml volume of your diluted antibody and incubate on a shaker slowly to ensure your whole membrane is always covered. Alternatively if you have a roller available I put my membranes in a 50ml tube containing 5ml of my diluted antibody and have to rotating to ensure coverage of my membrane. From experience there is more chance of issue with even staining with antibodies when using the pouch method.

Make sure that when you are incubating your ECL that your membrane is protein side up and that there is enough volume so that it does not leave dry patched on your membrane, keep an eye on the solution to make sure that during the whole 5 minute incubation it is not draining to one side leaving the other side to dry out.

I can offer a detailed protocol if you like.

Hi Devon

I agree with Elisabeth. Multiple stripping can denude some of your protein, especially when you strip at 50C which is quite agreesive but admittedly sometimes necessary especially with strong initial signal and high background

A couple of things to add:

1. You could try blocking over night @ 4C instead of 2 hours. Sometimes this helps

2. I note you are using PVDF membrane which is more durable than nitrocellulose/protran and bettwer therefore for stripping. However, it does suffer from higher background issues than Nitrocellulose/protran and is less sensitive, which could increase your background. Thus you could try switching your transfer material from PVDF to Protran/Nitrocellulose

Regardless, I tend to probe for my GOI, strip and then re probe for my housekeeping gene to check normalisation

Multiple stripping for a panel of different genes of interest is not a good idea. For each gene of interest perform individual western blots and try and strip and reprobe just to verify normalisation of your samples with respect to a housekeeping gene. Successive stripping will definitely increase your background, especially using PVDF

You might also want to double the amount of Tween 20 in your TBST and was for upto 6 x 5 min instead of 3 x 5min. This can also on occasions help

Finally, keep in mind that specific signal and signal to noise irrespective of the above is very much a function of antobody provenance: an antibody from one company will yeild poor signal to noise and thus high background whereas an antibody from another company even raised to the same sythetic peptide will yield a much cleaner blot (presumably this has something to do with immunisation protocols and subsequent antibody harvest)

See some attached materials including prior answers on this forum relating to your issue

https://www.researchgate.net/post/How_to_reduce_background_in_Western_blot

Hope this helps !!

http://www.abcam.com/protocols/western-blot-troubleshooting-tips

Hi there,

The  western background trouble  :

My recommendation to troubleshoot your problem :(to reduce nonspecificity)

1. Use monoclonal antibody, in case if you are using polyclonal derived antibody. 

2. Increase your blocking percentage of BSA/CASEIN .

3. Increase the detergent percentage such as Tween 20 or Triton X to break the weak interaction of the antibody.

4. Use minimum substrate luminol and H2O2 should be equally spread . 

5. In washing step use fast rocking. 15 minute each (3X)  at primary wash and secondary wash.

5. Play with exposure and sensitivity of the gel doc in which you are acquiring your image. if required.

All the above suggestions are directed towards the concern of non specificity.

Hope it was helpful

Rajkumar Dhanaraju , 

Hey Elizabeth, 

Thanks for the advice. I would really appreciate your protocol so I can compare and contrast what I have on hand.

Two additional questions:

-Do you add any SDS to your transfer buffer?

-What voltage do you typically transfer at and for how long?

Hey Laurence and Rajkumar,

The TBST I am currently using is .05% Tween. Should I increase to .1% tween?

Devon, I have attached our WB protocol. It is very descriptive as it is used to instruct new students.

Hi Devon

you can actually go much higher than that but try 0.1 to 0.2%. I think your main problem is recurrent stripping and reporobing plus use of PVDF which will exacerbate background

Others have provided a lot of useful information about Westerns. I would simply add that it helps to always probe first for your weakest bands, because they will be easiest to strip and at the same time, will be most degraded by stripping. A loading control like GAPDH I would typically do last, for example. Also, to avoid excess stripping, I routinely run duplicate samples so I have 2 identical membranes to work with. And one more thing- your stripping protocol seems fairly stringent - you probably do not need this degree of stripping for every band.  

Hi Devon,

Regard to your question on percentage of tween. You have to take the trouble of standardizing with increase concentration of tween . Your Wash time and fast rocking is critical.

To add to my original Elisabeths and specifically Diana's comments I would follow and indeed do practice Diana's advice: western blot for your gene of interest, strip and then re probe for your housekeeper; not the other way around. Then as mentioned perform a new blot with a new gene of interest. This is because it is easy to strip away nominal signal from your gene of interest compared to very strong signal from very high copy number housekeeper. Indeed it is not always possible to completely strip housekeepers  so choose a housekeeper that is at least 5kd different in size from your gene of interest. I will conclude with 1 final comment: in order to remove a housekeeping gene you need to strip aggressively at high temp. This will inevitably denied some of your other protein including gene of interest and since housekeeper is not always completely removed this can and often does distort the answer you get from a quantitative western blot. Thus for completely quantitative westerns in particular probe with gene of interest strip probe with housekeeper then prepare a fresh gel for your next gene/ antibody. Do not attempt to strip your blot after housekeeper and then re probe for a second gene