Hi Jatuphon,

I suspect that antibiotics will be even less stable than antibodies; pasteurising is probably an option, but once you put your diluted antibody on a blot, its effect will be lost.

The two main options that I came across are:

(1) freezing the diluted antibody between uses (which I personally don't like), or

(2) adding Sodium Azide to a final concentration of 0.02-0.03%. Sodium Azide is a very potent preservative, I think by inhibiting bacterial cytochrome oxidase, but I could be wrong. I have successfully kept diluted antibodies in the fridge for months, and re-used them until the signal faded due to antibody depletion, or the milk separated into such an ugly state that it was aesthetically offensive... :-)

BEWARE 1: Sodium Azide is very toxic, and all stock solutions (mine was 3% w/v, used as 100x) should be clearly labelled as such.

BEWARE 2: Sodium Azide will completely inhibit HRP, which is the typical coupled component in secondary antibodies that you detect by chemiluminescence. Wash the blot at least 3 times after incubating with the preserved primary antibody (which you should do anyway - no need to be extra paranoid), and NEVER preserve your HRP-secondary with sodium azide. I have done it, and you get very very clean autoradiograms.... :-)

Hope it helps.

Good luck,

Anna

Thank you very much, sir.

According to this http://biology4.wustl.edu/levin/protocols/antibodies/Antibody-storage-reuse-Western-pdf.pdf, is 0.02% NaN3 ok?

Hi Jatuphon,

I suspect that antibiotics will be even less stable than antibodies; pasteurising is probably an option, but once you put your diluted antibody on a blot, its effect will be lost.

The two main options that I came across are:

(1) freezing the diluted antibody between uses (which I personally don't like), or

(2) adding Sodium Azide to a final concentration of 0.02-0.03%. Sodium Azide is a very potent preservative, I think by inhibiting bacterial cytochrome oxidase, but I could be wrong. I have successfully kept diluted antibodies in the fridge for months, and re-used them until the signal faded due to antibody depletion, or the milk separated into such an ugly state that it was aesthetically offensive... :-)

BEWARE 1: Sodium Azide is very toxic, and all stock solutions (mine was 3% w/v, used as 100x) should be clearly labelled as such.

BEWARE 2: Sodium Azide will completely inhibit HRP, which is the typical coupled component in secondary antibodies that you detect by chemiluminescence. Wash the blot at least 3 times after incubating with the preserved primary antibody (which you should do anyway - no need to be extra paranoid), and NEVER preserve your HRP-secondary with sodium azide. I have done it, and you get very very clean autoradiograms.... :-)

Hope it helps.

Good luck,

Anna

Hi Anna,

This is very helpful!

I'll carefully handle Sodium Azide.

By the way, how many time can we re-thaw our diluted primary antibody?

And, is there any short way to examine reused primary antibody before use since I don't want to lose the sample?

Thank you very much,

Jatuphon.

I cannot comment on freeze-thawing as I have always kept my diluted primaries in the fridge. With simple re-using the decline in signal is very gradual, so you are unlikely to have a sudden failing Western - your exposure times will gradually increase, that's all. In any case, if you find that the signal has gone too low, over-probing with a fresh dilution is perfectly OK. I am not aware of any simple method to test the antibody before use, and I honestly don't think there is a need to in most cases. If you are probing for exceptionally abundant proteins (e.g. yolk protein in oocytes), the depletion of the antibody may be quicker, but this is so antigen/antibody-specific that any generalisations are bound to be misleading.

Good luck,

Anna

IMHO milk is not a good medium, the shelf life is not good.

Try adding sodium azide to it.

Bovine Igs stays active for two months in out experoments when milk was sterile:

Wei et al. 2002. Stability and activity of specific antibodies against Streptococcus mutans and Streptococcus sobrinus in bovine milk fermented with Lactobacillus rhamnosus strain GG or treated at ultra-high temperature. Oral Microbiology and Immunology 2002; 17, 9-15.

Antibodies will stay active for 10 yrs if you lyophilize the skimmed milk and store the powder in dry at +4C. Bovine antibodies tolerate also renneting soyou can precipitate caseins with chymosine and lyophilize the whey fraction to obtain higher Ig concentration? The sugars can be removed by nanofiltration or chromatography.

add sodium azide...it inhibits bacterial growth and prolongs antibody storage in milk....

If you can do this, don't dilute you antibody in milk. Even with the Sodium Azide, the milk will eventually curdle. Several years ago we switched to diluting all of our antibodies in 3% BSA/PBS with 0.02% azide for use in Westerns, imaging etc etc. It keeps forever.

I agree with Bryen milk itself is very fragile... We generally used antibody diluting solutions. When I need huge amounts of dilutions I even used PBS+Tween with sodium azide, it worked and lasted very well for me... But I apply freshly prepeared 2% skim milk incubation just before protein blocking...

The best way to store antibody is in plain buffer without skimmed milk or BSA. I always use antibody without mixing with skim milk or BSA and they work well with no noise. If your blocking is good then i dont think there is a need to use skim milk in the following..

The best way to store antibody is in plain buffer without skimmed milk or BSA kept in 4 degree (if to be used imediately or -20 if you wana store it) I always use antibody without mixing with skim milk or BSA and they work well with no noise. If your blocking is good then i dont think there is a need to use skim milk in the following..

Try gentamicin sulfate at 50 ug/ml.

Thank you everyone for your suggestion.

Right now I'm using 3% sodium azide and hence my antibody's lasted up to a month.