Hi, Atta, you asked very interesting question!. Really, in the WB you can select specific band based on protein size, but also intensity. I am not sure you can use same trick in immunolocalization, espesuially if in WB you have multiplay bands with similar affinity (intensity).

However, in immunolocalization you may use the otrher way: if you know protein localization (nucleus, membrane, mitochondria), you canm choose "right" signal according to localization. Moreover, you can alwasy regulate affinity by alter conditions of antibody incubation, as well as during the washing by increasing detergent contents in the washing buffer. But, generally, you can use only highly specfic antibody to 100% trust the resukts!

Good luck!

I agree with my previous speaker. Just some additions: you can also play with the dilutions of your primary and secondary antibodies. Im always using 1: 20000 in both cases. Another thing is to stop the incubation with your substrate as soon as you see your band of interest, when using alkaline phosphatase conjugates. Longer incubations lead to unspecific stainings.

One question: how do you block your blots, prior to application of your first antibody?

Hi Rahman,

To check specificity of a monoclonal antibody in immunofluorescence, you can check specific staining with increasing or decreasing antibody concentration or pre block antibody with antigen and then check!

Hi, Atta, please, do not forget about possible options as blocking peptide.

But, nay way, if you have 2 bands in WB with the similar intensity, you can not use Ab for immunolocalization.

I like this question a lot! Who is wondering now about the artifacts can be shaved on the picture, the mainstream now is to generate as much as possible… Not many young researchers is asking now the question which result shows the truth. My results of shaving Western-blot you can see here:

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

With antibody there is only one truth control, the negative one. Knock-out, knock-down, or the conditions described by the specialists as causing the down-regulation of your protein. The band on the proper mass on Western means nowadays, the company verified the antibody against many tissues, picked the one with the desired band mass. Many times happen to me it was an artifact…

It is always critical to choose the right negative control(s) - a genetics group I have seen, surprisingly collected their negative controls for immunohistochemistry to "develop" them seperately at a different day, i.e. they usually never included their negative controls in parallel in the staining of their tumor samples ... I was shocked but they did not change their practice.

And in some approaches the polyclonal serum can have advantages over the monoclonal antibody.

Another group tried to do some difficult stainings (including immunofluorescence, immunocytochemistry) for obviously difficult to detect interleukins, but failed for many years trying any method and trick available ...

So, I hope you will choose the right controls. Good luck.

Thanks to all of the responders with fruitful suggestions. Since the point is to reduce the cross reactivity of monoclonal antibody with mono-specificity, I suggest to do it by optimizating the working condition especially finding the right dilution of the antibody to recognize its antigen. No doubt that approperiate negative control and isotype match control should be included.

Dear Atta,

we have evaluated the specificity of our monoclonal antibodies by surface Plasmon resonance Analysis against related Proteins/peptides or truncated forms of the target Protein. This technique gives you much more Information than any others. You can calculate the Ratio of cross reactivity and can test also conditions to supress it. Requirment for this Analysis is your target or off-target in purified form.

If you Need further Information, send me an answer.

Best wishes,

Martin

There are more than one way to attack this problem specially in WB. One way is to titrate the amount of your antibody because in lower dilution of antibody your going to lose the nonspecific binding and retain the specific binding. Th second way is to have very good blocking step with 5% milk or 5% BSA to prevent the non specific binding. Also include 2.5% milk with your primary and secondary antibodies. A Good washing step after your primary antibody. My advice is to combine all approaches. For IF I think the titration will help, but you have to confirm your results with another mean e.g. another antibody or the localization of your protein.

Another idea is to include a control non-relevant monoclonal of the same species and isotype to check for non-specific binding. If your test monoclonal is not labelled, I assume you already include a secondary antibody control.

Good luck

1. Analytically speaking, if the epítope recognized by such monoclonal antibody is moderately represented in negative controls, concentrations could be managed in order to significantly discriminate the positivity. Eventually new monoclonal antibodies need to be generated to identify an exclusive epitope not existing at all in negative control or non-relevantly represented in order to manage the positive/negative relation in the analytical assay

2. If the goal if immunoaffinity purification the quantity and quality of the possible contaminates need to be evaluated in terms of chromatography yield and overall performance

best regards and good luck

Can you do the immunofluorescence minus and plus added peptide that is the epitope for the Ab. If the peptide competes away the signal, then you know it is specific.

You can do any detection technique with a peptide that competes for binding with the same epitope as your Ab. Tara's is this what you meant by a blocking peptide?

I second Dr Kamaleldin Elagib's answer. lowering dilution and an additional blocking step combined with a mild detergent included wash solution will definitely improve the specificity.

Unspecific binding can be due to the secondary antibody.

If the problem is caused by your primary antibody, you may saturate unspecific binding sites using a blocking antibody (that is not recongnized by your detection antibody).

As mentioned in the very first comment, if the antibody is used in immunofluorescence for structural studies, non specific fluorescence can be very easily distinguished from specific fluorescence from the size, shape & location of fluorescence in the cells/tissue. One has to observe carefully & learn to make this distinction!

It is not uncommon for a monoclonal antibody to have multiple, apparently unrelated binding activities. This is not due to conventional cross-reactivity to structurally-related epitopes but is more likely the result of binding by non- or partially-overlapping regions within the Fab of the antibody. As a result a MoAb selected for one reactivity my have accidental activity to an unrelated epitope. There are several examples of this in the literature going back some 30 years. So it is possible for a MoAb to have high avidity reactivity to two unrelated antigens that would not compete with each other. The only way around this problem is to try another MoAb to the antigen of interest as Dr. Otero-Gonzalez suggested, or polyclonal antibodies may work better as Dr. Eibl suggested because any antibody multireactivity of constituent antibodies would probably be diluted out by the other antibodies in the mixture. If MoAb multireactivity is the basis for your problem, “optimizing the working conditions” or blocking with soluble antigen will probably not help and an isotype-matched negative control does not really address this problem.

Since the mAb recognizes just a small specific sequence, it makes no sense to compete with a peptide, as obviously, it recognizes this or a related sequence in other proteins. Your best bet is try to screen several mAbs for least background in IP/WB. Anyway, this has always been my biggest issue with staining and immunofluorescence studies. How do you control for possible cross-reactivity. At least on blot you can see different sizes and exclude these by size. In addition, if your protein is regulated (degraded or upregulated by signaling events) you can also distinguish this. But really, how can you be certain in FACS or immunostainings whether or not you are only targeting your protein of interest. Even if you do a WB and you see just one band, it doesn't tell you whether your mAb reacts with other NATIVE proteins. I guess the closest to checking whether your protein is specific in native conformation, is a pull down with the antibody and check purity on coomassie/silverstain gel but still that has many issues as well. I guess for me it would be more convincing if you could show colocalization with a known interaction partner. I guess, you can only do so much to convince yourself and others...as for a comment on being able to see 'non-specific' staining in immunostainings.. there is no such thing as non-specific fluorescence from the mAb... it binds very specifically to the target sequence... it just happens to be in multiple proteins... if you were talking about some sort of interactions due to other domains of the mAb, then it is a different story...

An isotype control mAb is good for sure, but this does not reveal if you mAb has binding capacity against other proteins. If it has so, but with lower affinity, you need to dilute the mAb. Incubating with immunized antigen- peptide/protein is a good control to test specificity.

Also, if possible, use different tissues, e.g. a knockout strain or tissue phenotypes, or conditions wherein the protein is not expressed/overexpressed.

Many commercial (and also all other human and murine) monoclonal antibodies recognizing not a single antigen only. Some of them recognizing not only related epitopes, some also reaction with antigens like DNA, LPS, ....

It's described a long time ago, but it's something you need to have in mind (Curr Opin Immunol. 1995 Dec;7(6):812-8. Natural autoantibodies. Coutinho A, Kazatchkine MD, Avrameas S., Allerg Immunol (Leipz). 1990;36(3):163-77. Multireactive human monoclonal antibodies. Kiessig ST, Jahn S, Hiepe F, Volk HD, Fietze E, Zugehör M, Porstmann T, von Baehr R.)

If you see those phenomenons, you have to think about this. You can check the multireactivity of antibodies simply by immuohistology on different organs. Here you have a large set of antigens present at the same time.

To overcome this issues, check several available antibodies until you find one working.

Specific fluorescence can be identified from non specif. However, use serial dilutions to confirm. As mentioned by [Neelima Deuskar], one learns from careful repetitive observations to distinguish. lalitha kabilan