Maybe you can test your antibody making a dot blot. It represents a simplification of the method. In a dot blot the proteins to be detected are not first separated by electrophoresis. Instead, protein sample is spotted through circular templates directly onto the membrane or paper substrate. Protein samples are not separated electrophoretically. This is then followed by detection by antibodies. This technique offers savings in time although offers no information on the size of the target biomolecule but you can use different antibody serial concentrations to check if you antibody does work or not.

Hi, you can do Coombs test for testing an antibody. The principle behind this method is integration of your intact antibody with blood cells because red blood cells are typed with certain antigen on the cell surface. Usually antibody considers it as a foreign agent and causes destruction of blood cell you may see agglutination of red blood cells.

I will suggest you just try the WB. Do not even trust the manufacturer's website info. I have used a few expensive antibodies form trustworthy companies and was not able to see anything or just obtained terrible background signals. There is really no short cut if your research will rely heavily on that specific protein.

Maybe you can test your antibody making a dot blot. It represents a simplification of the method. In a dot blot the proteins to be detected are not first separated by electrophoresis. Instead, protein sample is spotted through circular templates directly onto the membrane or paper substrate. Protein samples are not separated electrophoretically. This is then followed by detection by antibodies. This technique offers savings in time although offers no information on the size of the target biomolecule but you can use different antibody serial concentrations to check if you antibody does work or not.

I also suggest to test the ab by WB, since at the end you would like to use this methode. Dot blots have the disadvantage to use the antigen in a more native state compared to the denatured state you would use in a WB. You could test the antibody using a recombinant antigen for sensitivity and a crude homogenate for specificity before you use your more important samples.

measure your antibody and proteins separately and them incubate them for 15 minutes at 37 then pass it through 10KD membrane and again measure the quantity. It should tell you whether your antibody bound to your protein or not.

Sorry, if your protein is more than 10 KD in size then you have to use bigger size membrane which will allow your protein to pass smoothly and for control you can simply pass your protein with same concentration.

There is no shortcut for antibody testing as per of my knowledge

Testing an antibody depends on the application you want to use at least. So if you will analyze more native proteins you may use Dot Blot as a fast way. If proteins should be analyzed as denatured samples you may use WB as the best method. But if the aim is to identify proteins in cells or tissue sections then it is absolutely necessary to use immunohistochemical protocolls to test the antibody. Successful antibody binding to his antigen is often not comparable between these applications (Dot Blot vs WB vs IHC).

Methods from the dim dark past: Use an Ochterlony double immunodiffusion plate (http://en.wikipedia.org/wiki/Ouchterlony_double_immunodiffusion). Its very simple pour an agarose plate then punch 2 small holes in it. Put antigen in one hole and antibody in the other. If the antibody is working you will see a white precipitin line forming.

Two words of caution:

1) I do not think it works for monoclonal antibodies.

2) And some rare proteins do not necessarily form insoluble precipitin

Dot blots and ELISA's are alternate options to check an antibody. Having appropriate negative controls is critical if you choose these quicker methods. If you are planning to have a career in the biotech industry at some point, it would be advisable to learn how to screen antibodies with ELISA assays. ELISA assays are core QC requirements for many companies and Westerns are often not considered acceptable due to lack of quantitative data. There are new automated Western replacement technologies such as http://www.proteinsimple.com/simple_western_assays.html These technologies may eventually replace Westerns. Let us hope so. After doing hundreds of Westerns over the years, I can certainly understand why you are looking for other protein detection options.

There also some other modifications of the dot blot/ELISA, based on thier simplicity (without electrophoresis) that allows you to test several antigents in a simple way, is called MABA (http://www.sciencedirect.com/science/article/pii/S0165247898000558). And as Jeffrey said, is always advisable to know how to perform ELISA assays.

If you don't want to do a western blot then you should reconsider working with antibodies. Some things are just essential. WBs tell you not only the reactivity, but also the size of the protein and whether or not other proteins, i.e. other bands , react with your antibody. As said above, Just do it!

There is just a simple short cut: a dot-immuno assay. Use the same nitrocellulose (or any other membrane of your choice) and pipette spots/drops of different antigen-concentrations (5 µg/mL .... 0,001 µg/mL) with a volume of 0.1 to 1 µL. To simulate the SDS-concentration during electrophoresis compare SDS- vs. non-pretreated samples used for spotting.

The binding of your protein onto the hydrophobic membrane surface will happen within a short time (up to 2 h). To avoid effects caused by drying, incubate your membrane in a wet chamber.

After this step, wash the membrane strips with PBS 0.1% v/v Tween 20 4 times and incubate the strips with the antibody in question. All other detection steps (anti-species-enzyme labeled, color reaction) are well known. You can compare the sensitivity of your antibodies by the detection limits on the different membranes.

Peronal experience and an update as below

1. Have tried dot blot (to quantify total amount of signals) numerous times with help from experienced hands but never worked for me. Maybe I am just clumsy to it but my experineced colleagues could not figure out why. But I was able to get reliable signals by WB.

2. Sometimes the membrane can give you different results. I just did one this week with same amoount of protein loaded on both PVDF and nitrocellulose. Amazingly one without any signal for the protein of interest after using femto ECL whereas the other showed good signal with pico ECL. After working with WB for 18 years this is my first experience which I have no clue why.  

Monoclonlas have to checked according to their later indications.

General checks are:

1. Recognized antigen by direct binding and competition assays

2. Check with (potential) cross-reactive antigens (antigens with known relations, with known identical peptide sequences

3. Affinities to the target antigen (and to the cross- or mutireactive antigens as well)

4. Check for multireactivity (see attached reference)

5. You need to know something about the epitope (native, non-native, linear peptide sequence, …)

6. ELISA, FACS, immunohistology, Friguet’s Affinity detection, Western Blotting, PepScans, PlasmonResonance, several in-vivo-assays, complement binding, … there are many methods available.