Good day!
I've found many different western blot protocols and it is mostly recommended to prepare primary antibody hybridization solution with blocking buffer (5% skim milk in TBST) and to discard it after 12 h. In other protocols it is mentioned to use wash buffer (TBST, or simply TBS), and also discard after 12 h.
On the other hand, it is mentioned by the manufacturer that antibodies are stable in sterile water for 1 month at +2 +8C.
I've tried to use the water solution (1:1000) for hybridization - it works fine. All the manipulations were conducted in a laminar box. But after 1 week at 4C antibodies seemed to be precipitated - there is a slightly pink precipitate at the bottom of the tube. The supernatant is clear. Can it be reused after resuspension? Maybe there is a way to improve solution stability?
Your antibodies are likely to remain stable at 4oC if diluted in a BSA/protein solution BUT you will need protease-free solutions and microbial inhibitors to get extended fridge life. Exactly how long is going to be highly antibody dependent. You'll need a positive control on each blot.
What I find bizarre is the suggestion from the manufacturer that you can keep antibodies in sterile water. I can only imagine they are suggesting that you add sterile water to a freeze-dried antibody, hence the buffer salts are provided by the antibody sample? Pure water is risky as it is far removed from the conditions in which antibodies normally work i.e. isotonic conditions.
Add a little sodium azide to about 0.1% m/v to keep microbes at bay. Be aware that it will deactivate HRP, though. So you need to remove traces by washing well with PBST before you incubate with the secondary AB.
When you use water only as diluent you're risking to lose significant amounts of antibody by adsorption to the container walls. Add at least some non-ionic detergent (e.g. Tween 20) or better also a carrier protein, e.g. BSA or gelatin, which also then will keep possibly present proteases rather occupied.
Best to think of the diluted antibody solutions as of limited life/reuse and to remake this for critical purposes. However some limited reuse it possible when eg bsa/skimmed milk ( to prevent ab sticking to the container) and eg azide antimicrobial are added.
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