Manufacturer declares the applications that have already been tested. It doesn't mean that a given antibody is not useful for other applications, you can test the one you need. But have in mind that not every antibody works in any experimental system. For instance.

Antibodies for FACS need to recognize an epitope exposed when the antigen is displayed at the cell surface. Alternatively, if the epitope is located in the intracellular tail of the molecule, you need to perform permeabilization before staining.

Antibodies for IHC should recognize the antigen after tissue processing, for instance with organic solvents used for fixation on slides and paraffin embedding. Some antibodies only work on fresh tissues.

Neutralizing or blocking antibodies have to recognize epitopes that are close enough to the antigen regions involved in the interaction of interest. Otherwise, they would not block the biological function.

ELISA: protein expression (tissue or cells)

FACS: fluorescence expression protein levels (tissue or cells)

IHC: tissue sections expression levels

Manufacturer declares the applications that have already been tested. It doesn't mean that a given antibody is not useful for other applications, you can test the one you need. But have in mind that not every antibody works in any experimental system. For instance.

Antibodies for FACS need to recognize an epitope exposed when the antigen is displayed at the cell surface. Alternatively, if the epitope is located in the intracellular tail of the molecule, you need to perform permeabilization before staining.

Antibodies for IHC should recognize the antigen after tissue processing, for instance with organic solvents used for fixation on slides and paraffin embedding. Some antibodies only work on fresh tissues.

Neutralizing or blocking antibodies have to recognize epitopes that are close enough to the antigen regions involved in the interaction of interest. Otherwise, they would not block the biological function.

Hi Yoorha, how are you doing today?

Well, there is no difference in antibodies for ELISA, FACS or IHC. Actually, the same antibody can be applied for all techniques. However, you need to adjust the antibody correctly for each one. The antibody target one epitope in the protein, this epitope can be presented in different ways in each technique. In ELISA and FACS, we usually work with live cells, while in IHC the tissue or the cells are fixed. In ELISA and FACS there is no alteration in proteins, while in IHC generally the protein epitopes are masked by the fixation process and you need to unmask the epitope for antibody detection.

Considering that the antibody does not be conjugated, you need to use a secondary antibody conjugated with some revealer. Secondary antibodies usually are conjugated with fluorescence dye or enzymes such as Horseradish peroxidase (HRP) or alkaline phosphatase (AP). In ELISA and FACS, most secondary antibodies have fluorescence dies, while in IHC the enzymes are more routine, but you can use fluorescence dyes in IHC with no problem. Commercial antibodies have described in data sheet what application they were developed for. You can buy an antibody and test in other applications but you have no sure if the antibody woks in techniques that were not tested before.

You can use antibodies in cell culture to block certain molecules. However, I think this work only in membrane proteins such as receptors or binding proteins and proteins in the cellular microenvironment. Intracellular proteins you need to permeabilize the membrane to allow the antibody enter in the cell. Also, you need to perform cellular membrane permeabilization in ELISA and FACS to detect intracellular proteins. But when we do that, we usually use a permeabilization with fixation kit. I never use an antibody to block intracellular proteins, so I do not know for sure about that. My best regards and good luck in your research.

Hi Yoorha

Antibodies are the same for these techniques. Most of them are classical IgG raised in mouse or Rabbit. You could find some specific smaller antibodies from llama which may be usefull in fine staining when performing tissues immunohistochemistry, due to better stability and capacity to detect hardly accessible proteins. Derived from the later, even smaller nanobodies also exist which can be directly expressed in cells by molecular engeneering and avoid double classical coupling.Not sure all these can be applied to your research topic but hope it helps you.

Good luck

As others already stated, not the antibody itself but the epitope presentation plays the major role as they are influenced by the different techniques. And the presentation of the immunogen during developement of the antibody already determines some binding properties.

Antibodies working with aceton-fixed cryosections might not work with HCHO-fixed paraffin-embedded sections. Antibodies for Western Blotting e.g. react against mainly unfolded, denatured antigens as the results from reducing SDS-PAGE. And intracellular antigens in cell assays have to be accessible for the antibody.

It's always a trial-and-error process to find out if an antibody work for your purpose, especially because besides pre-treatment of the antigen also concentrations and incubation conditions might also play a role.

Therefore it's worth also checking literature what others already have done with a specific antibody or if there's an antibody already working for your purpose.

Good luck!

The same Ab can work for all but it depends on several circumstances as :

1- sample type " cells, tissue, extracellular protein, nuclear or cytoplasmic protein" which will directly affect the Ab-protein interaction.

2- The Ab Concentration, Penetration, Stability as well as Specificity. all of this can limit the number of the techniques that can be validated with this Ab.

3- Availability of the secondary Ab that work 100% with each technique.

4- What do you want to detect and when as a kinetics, This is became an a new important advice to be taken in your consideration when choosing a technique.

5- Making sure that you have the right resources and instruments as recommended by the company's.

6- Your work flow and the instructions as well as the analysis : sometimes researchers don't follow these correctly which is sometimes lead to wrong analysis and interpretation of the data.

7- I have seen it myself the type sample matters a lot, like working with Adipose tissue is totally different from other tissues. as well as small particles like Exosomes.

8- You have to go through a very well controlled optimization for each technique.

Hopefully this can help.

Good Luck

Hi Yoorha,

Concerning your last question, you should look as cancer immunotherapy techniques which may be give you some ideas.

Good luck

You choose your antibody depending on the experiment of choice and nature of your protein of interest. For Elisa you basically need an antibody that targets a native protein ( epitope on the 3d structure of the protein) example serum proteins. For FACS or IF you also need your ab to target native structure. Only difference in FACS you need suspension cells or trypsinised.. IHC is same as IF uses native protein but embedded in tissues fixed and sliced using microtome. Western uses denatured proteins so your ab should can target any seq. Using antibodies you can only target proteins. If you want to block a protein on cell any ab that targets the native structure works. Chemicals cannots be targeted unless you have a conjugated partner tagged on ur ab.

The main difference in the antibodies for different application is concentrations. The antibody for IHC is highly concentrated (as you need to dilute it 100-1000 times before use), you may use IHC compliant antibody for ELISA and FACS too.

Antibody concentration of ELISA is in few "X", so need to dilute it few times (2x-50X), you may use the antibody for FACS but it may not work very well for IHC.

FACS application antibody comes in ready to use concentrations. These are the most diluent one. If you do IHC with it, you will merely get signal.

Then come stability of antibody. Antibody for different application is tested against the reagents to be used in process. FACS antibody may not work with IHC as this may not stand with organic reagents used in the process.

Lstly to your last question; Yes you may use any of these antibody to block the paratop in culture system.

What I understand is there's no difference. But all need to be optimised for specific experiments. Before use.