for secondary antibody, I usually dilute it in TBST with 5% skim milk (weight/volume) and store them in a -20C. I reused them for up to 8 times and bands were clear.

for primary antibody, I recommend using BSA for they can stabilize antibodies and proteins during freezing process

some antibodies should be stored in 4C not -20C you may find this information on the product sheet provided by Ab supplier

Best Regards.

Thanks, Donghan Kim for the help.

Could you please tell me what % of BSA should be present in TBST for the storage of primary antibodies at 4°C?

Hello Saurabh Tiwari

The primary antibody may be stored at 4 degree C for re-use on later blots. But the biggest risk in reusing primary antibody is microbial contamination. To avoid unwanted microbes, you may add sodium azide (0.02%). Although most antibodies are packaged with sodium azide in the buffer, there’s not enough to be effective in larger diluted volumes. But the problem is despite the addition of sodium azide, milk eventually will go bad. As an alternative, diluting in 5% BSA solution is a good option. BSA functions as a protein stabilizer, allowing you to re-use antibody longer when stored at 4 degree C.

If you need to re-use your antibody over an indefinite period of time between blots, you may make up the dilution of your primary antibody in skim milk, without sodium azide, and freeze it at -20°C. BSA is not as effective for protecting your antibody from the freezing process as milk is. Another issue of storing the primary antibody in -20 degree C for re-use is repeated freeze-thaw cycles, which can denature the antibody. You need to take a note of this.

Best.

Hello!

Primary antibodies diluted in just TBST can be recycled and stored for later use at 4 degree for 3-5 times.

Best

Hi Malcolm Nobre and Haroon Khan

Thanks for your responses!

Regards