I do not have any experience in freexing 'mixes' but I have routinely used frozen primary antibody made up in blocking milk and it has worked. That said, yes, there are limitations to the number of times you can freeze-thaw the aliquots and the antibodies do lose their affinity/potency after repeated freeze-thaw cycles. Hope this helps.

It depend of the antibody.

Antibodies coupled to enzyme should not be frozen.

Antibodies coupled to fluorophore have to be frozen and keep on dark.

Ig3 antibodies does not support freezing because they form aggregates

Well mixing both antibodies may gives you unusual background(if secondary Ab binds any space of the blot); and freez thaw cycles do reduce the activity(binding) of Ab(s). That is why some antibodies(which loose it stability during freeze thaw cycles) are preferred to keep 4degrees always(in presence of 0.05-0.1% sodium azide to prevent any microbial growth). So follow the suggestions provided in the company data sheet. Freeze the Ab, if it is OK, and use untill it gives you nice signal.

We also do the same that Edward here

For us, the freeze thaw cycle works quite well, for several number of blots nicely with many antibodies(diluted Ab in milk), so it is a standard way in our lab.

Usually I will do it for an antibody that gives you a clear band, you can do it several time but I will not exceed to 10 times. Regarding the secondary antibody actually I have never done it, usually they are not so expensive and you use them highly diluted, I will not recommend.

Commercial antibodies have azide which stabilize the antibody longer. And most of the antibodies you can keep in 4C. Also it depends on isotypes of the antibody. IgG1 is the most stable antibody. You can freeze thaw but if you do a lot, it definitely looses its binding affinity significantly. I would prefer to use as aliquots.

If antibodies are allowed to keep at 4C, you can use years.

I think that it depend of the antibody. The IgG1 is the most stable antibody, and IgY is the unstable antibody during freeze thaw cycles.

It depends upon how strong is your Ag-Ab reaction. For example, for some antibodies like anti-alpha-A crystallin MAb which shows a very strong reactivity across species for the lens alpha-A crystallins; it is really possible to reuse the antibody mix several times, can be 5-7 times. However, in other cases, when the Ag-Ab reaction in the first instance itself takes several minutes to light up a band, reuse of the mix has no meaning.

Most of these answers are correct in some way. Concentrated antibodies are more stable than a dilution. However, if they are diluted in a blocking buffer like BSA or serum then the dilution has little effect. Ellen He is correct also that the isotype of your antibody will be just as important. For example in general you should not freeze IgM more than once but mouse IgG1 can undergo numerous freeze thaws or better yet keep it at 4C with .02% azide and reuse up to 10 times (for some high affinity antibodies like the anti-actin from Sigma A5441). Also consider that some antibodies come diluted in 1mg/ml albumin as a stabilizer for freezing. Another option would be to make up your antibodies in a 20-50% glycerol solution so that you can store at -20 but it wont form ice crystals. To some people this is the best of both worlds but I am not a fan of pipetting very small amounts of glycerol stock as accuracy suffers from the increase in viscosity. Lastly, if you do re-use antibody or freeze/thaw you should quick spin the tubes before making a new dilution or re-use to remove any precipitated protein or gunk from previous blotting. This debris can cause background and "mystery" patterns on your blot that leave you scratching your head in lab meetings! Good Luck.

Also you can aliquot your sample in small Eppendorf tubes and thaw only required number of tubes at a time. Thus you can avoid repeated freeze-thawing of the whole sample.

I prefer to keep Abs (diluted in BSA + Na azide 0.2%) at 4°C and reuse them for max 5 times. Froze and thaw cycles do not work fine!

I used to freeze thaw diluted primary antibody and re-use up to eight times before it stopped working - like so many things it is dependent upon what you are using and you just have to give it a try. I wouldn't freeze thaw secondaries, particularly those conjugated to HRP as you will find the HRP activity dies off, I have no experience with alkaline phosphatase conjugated secondaries, though I would assume the same is true of them.

It's may be possible but ...

antibodies in low concentrations as they are used as primary or secondary antibodies in blotting experiments are not very stable. For stable mixtures you need normally 1 mg/mL, for blotting you are using 1 µg/ml. Even if you are adding a stbilzing protein like BSA, you could never be sure if your antibody/antibodies are still working of if the antigen in you blot is not present.

Reuse is also not the best choice. First you are loosing some due to the immune reaction, second: the solution is not stable.

For a better comparability of your results, use a stock solution an for each experiment a fresh mixture.

for my work. the antibody that used will store at 4c but new antibody will stora at -20.. if you opened it... not recommend to keep it back to -20.. pls store at 4c.. it still well for several months.

Cheer

Oa

Be careful: some monoclonals are stable, others loosing their activity very fast.

I would like to thank everyone for your valuable comments!

Well. It's depends on the nature of antibody. From my experience, I kept diluted primary and secondary antibody in TBST at -20 C over few months. I also do freeze-thraw these antibodies and still can be used more than 5 times. However certain antibody may not work under this conditions.