Hello everyone,
I am encountering bacterial growth in my diluted western primary antibodies (in TBS, without any milk/bsa, with 0.01% NaZ). We keep the antibodies in +4 C since we use them frequently (Our incubations are also o/n at +4 C). Almost every 2-3 weeks I observe the contamination. I filter the antibodies with 0.4um filter every 1-2 months.
I am wondering why there is that much of bacterial growth even with NaZ. Also, is there a better way of decontaminating antibodies? Can I keep the antibodies in -20 C (how many times I can freeze/thaw them?)
Are you sure you're seeing growing bacteria, not precipitate of some kind? TBS alone can hardly support bacterial growth, and you even have it supplemented with NaN3. By the way, people rather use 0.01M or 0.1% NaN3 instead of 0.01%. If you are concerned, you may increase NaN3 concentration.
Apart from Sodiumazide mentioned above by Victor G Stepanov, people also sometimes use 5-15μg/ml of gentamicin as diluent for antibodies, but it has several cons such as: (1) antibiotic residues might affect downstream applications, particularly if used at high concentrations. (2) Some antibodies might be sensitive to the presence of antibiotics, potentially affecting binding or performance. (3) Resistance might develop in bacterial cultures over time, but if it works for the time you can see to it.
I have never experienced bacterial contamination in antibodies, nor know of anyone who has... Maybe a good thing to do (if you don't want to go through many freeze/thaw cycles) is to aliquot the undiluted antibody as soon as it arrives and keep these aliquots at -20? If you freeze/thaw an aliquot only a few times it should still be okay to use (I freeze/thaw my antibodies several times and they work fine).
Thank you very much everyone. I will start with Victor G Stepanov 's recommendation by increasing the amount of NaN3. I hope that will work.
ps: By the way, I am sure it is some sort of bacterial contamination with cloudy apperance and terrible smell :/
Carles Puig Saenz We actually aliquoted the antibodes when they are arrived. They do not have any contamination. The only contamination is in the diluted (in TBS) antibodies.
Yes, hence why it might be a better idea to aliquot the antibodies undiluted. :)
Come to think of it, western antibodies are often delivered dissolved in PBS or lyophilized from PBS. This would provide phosphorus for the putative bacterial growth, while Tris from TBS could be metabolized for carbon and nitrogen, and the rest could be taken from antibodies. Terrible smell from your antibody solution makes me think of some Bacilli, they usually grow very fast and stink a lot. So, it may happen that you have bacillus spores flying around, and they contaminate more or less everything but start to grow only after you dilute your antibodies with TBS, i.e. after they get all necessary nutrients in one pot.
Dilute your stocks in an equal volume of sterile glycerol and store at -20 C (1 mM NaAz optional). Keep in some kind of container (not an open rack) to minimize exposure to temperature swings for when the freezer door gets opened. Pipette what you need (doesn't freeze) and immediately return to freezer (don't leave in ice bucket).
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