Hi, I have not experience with DAB staining but your results seems to me the classical "ghost band", that, as Jurgen pointed out, were caused, generally, by insufficient blocking, proteins overload, excessive signal of primary/secondary antibody. In general with ECL you have white band on dark background, with DAB you have white band on "light brownish" background. You can find a lot of example, and explanation on the web for example:

http://openwetware.org/wiki/Griffin:Western_Blot_Optimization at the paragraph Ghost bands, reverse/white banding;

https://tools.lifetechnologies.com/content/sfs/brochures/TR0067-Chemi-Western-guide.pdf search for ghost band

/https://medschool.vanderbilt.edu/cdb-core-lab/files/cdb-core-lab/public_files/083013_WESTERN%20BLOTTING%20TIPS%20AND%20TROUBLESHOOTING%20GUIDE.pdf search for ghost band

In this case is important if the protein is abundant or not, because overloaded an abundant protein caused the problem. Could you try to load less protein, if it is not a problem to detect your protein of interest? I have a problem of ghost band with actin and I have to use primary and secondary antibody more diluted ( I usually use 1:2000 for actin) to have good signal.

I am sure you will resolve your problem!!!

Your photos show everything except the ECL to detect the HRP-linked secondary antibody. What is the purpose of the stain with DAB? I would understand if you stain your blotted nitrocellulose with PonceauS to see if proteins have been blotted...it should look a little bit like the Coomassie but pink instead of blue, and you can wash it away and proceed to blocking step, followede by antibody incubations and then ECL.

Regarding the white bands, you have a lot of protein loaded in each lane (I can see that from the Coomassie stain), and the most intense protein bands will completely block the nitrocellulose so there is no extra space for BSA. We tend to use milk powder in PBS-Tween20 as blocking agent, and it is possible that your 3% BSA blocks less than the actual proteins you have loaded in each lane. But I don't understand why you should stain with DAB. To detect your HRP-linked antibody, you need to do an ECL (using luminol, coumaric acid, peroxide and buffered at pH8.5 + X-ray film or sensitive digital camera to detect the blue chemiluminescence). None of your photos show an ECL result. 

Dear Jürgen Denecke,

Thanks for your answer. I used substrate DAB (Invitrogen) after incubating with HRP-linked secondary antibody. I did not do an ECL. So you mean to say that protein is overloaded and the blocking is insufficient ? And may be that's why secondary did not react with the primary or rather primary did not react to protein of interest because proteins are overcrowded ? 

And why BSA will block other proteins on the membrane ...it should block the unoccupied membrane ?

DAB provides a brown precipitate...it looks as if your protein bands have the least of this precipitate and appear white, whilst the part of the membrane that didn't receive proteins from the gel (but were blocked with BSA), are stained weakly and homogeneously. The stained parts were thus not completely blocked and have some secondary antibody sticking to it. The banding pattern in white looks very similar to the banding pattern on Coomassie, so these must be so abundant proteins that they block the nitrocellulose completely. But it doesn't look as if any of your proteins interact with primary antibody. It is basically a negative result and I don't see a positive control in your figures. I don't know anybody who uses DAB on westerns, it is more something people use in histochemistry. ECL is the most common way to detect HRP-linked antibodies on nitrocellulose.

Now, regardless of the detection method, to test if a western blot works, invariantly, you need a positive control (for instance an overproduced antigen in E.coli) and a negative control (E.coli without expression vector)... These two lanes need to be on every gel that includes unknown test samples, without exception. It makes no sense to load test samples (with unknown composition) and ask why it does not work when you have no controls. The positive control signal tells you that technically nothing stops you from detecting the antigen, and when your test samples don't show any signals, then your antigen is either below the detection limit or it is not there at all.  

Dear Sir, 

I had beta actin as a control. The first membrane showing up a single white lane is beta actin. Used mouse monoclonal primary and anti-mouse HRP secondary. And if it was a negative result beta actin should have showed up ?  Also, I have been doing beta actin regularly and I am sure nothing is wrong with anti-mouse HRP.  

So,, now if it's because of crowded proteins, at least, beta actin should have showed up considering the fact it is itself an abundant protein ??  I am really clueless ! 

Hi, I have not experience with DAB staining but your results seems to me the classical "ghost band", that, as Jurgen pointed out, were caused, generally, by insufficient blocking, proteins overload, excessive signal of primary/secondary antibody. In general with ECL you have white band on dark background, with DAB you have white band on "light brownish" background. You can find a lot of example, and explanation on the web for example:

http://openwetware.org/wiki/Griffin:Western_Blot_Optimization at the paragraph Ghost bands, reverse/white banding;

https://tools.lifetechnologies.com/content/sfs/brochures/TR0067-Chemi-Western-guide.pdf search for ghost band

/https://medschool.vanderbilt.edu/cdb-core-lab/files/cdb-core-lab/public_files/083013_WESTERN%20BLOTTING%20TIPS%20AND%20TROUBLESHOOTING%20GUIDE.pdf search for ghost band

In this case is important if the protein is abundant or not, because overloaded an abundant protein caused the problem. Could you try to load less protein, if it is not a problem to detect your protein of interest? I have a problem of ghost band with actin and I have to use primary and secondary antibody more diluted ( I usually use 1:2000 for actin) to have good signal.

I am sure you will resolve your problem!!!

Dear Dr. Gupta,

In response to your last message, let me define what I call positive and negative control:

You wish to detect a 17 kDa protein with Primary (Goat polyclonal (1:500) overnight) and Secondary (Bovine anti-goat (1:2500) for 1 1/2 hrs). A positive control would be a lane in which you load the purified 17kDa protein (or overproduced in total E.coli extracts). A negative control would be E.coli extract without the 17kDa protein. If you don't get a positive signal at the expected size in the positive control lane, and if that band is not totally absent in the negative control, then your experiment doesn't work.

The beta actin is just a loading control, it does not help you to interpret what you detect or don't detect with the anti-17kDa antibody, it just shows that you have loaded equal quantities of beta actin in the 5 lanes in the middle. The coomassie stain (or a PonceauS on the nitrocellulose) is actually better. Focus on the detection of the 17kDa protein first, and start with a dilution series of purified 17kDa protein if you have it. When you know the detection limit you can proceed with trying to detect the 17kDa protein within your 5 samples. Perhaps it is masked by a very abundant non-antigen of the same molecular weight which blocks the nitrocellulose completely. In that case, load less, try a different percentage gel, run the gel slower....but none of this is worthwhile if you don't start with a positive control of the 17kDa protein first and detect that.

Good luck, let us all know when you have solved the problem.

I have some experience with DAB stained Western Blots (s. attachment for an example; sorry for the quality, the blot is 15 years old, in the meantime we switched to ECL) and your blot is really negative. Follow the recommendations of Jürgen and you will find your protein, if it´s really expressed.

Thank you very much to all of you for your suggestions. Thank you Dr Jürgen for explaining in detail.  I will follow the above suggestions. I have done a few successful blottings with DAB for high molecular weight proteins and got good bands. I am having problem with small proteins. Anyway, I will do an ECL as well and let you know the results.