if antibody solution could be used again? - (Mar/24/2009 )

The first-antibody in blocking buffer (PBS with 3% BSA) and the second-antibody in blocking buffer (PBS with 5% nonfat dried milk), both of them were stored at 4℃ for one month, if i can use them for western blotting again? thanks.

As long as you don't see any bacterial growth, the primary antibody should be fine. If you do see growth (look for the pellet at the bottom of the tube) you may be able to filter the dilution and still have a usable dilution but the bacteria may have been able to lower the pH and destroy the antibody. I never save or reuse secondaries so I'm not sure how that will work if you try. If you want to save you primary antibody dilutions you should add 0.05% sodium azide. This will prevent the bacterial growth but kills the HRP on the secondary. Hence why most people don't reuse the secondary.

In fact, rather than stripping a membrane, I use NaAz (1% for 1 hour) if I want to reprobe with an antibody of a different species. This destroys the HRP (and signal) from the first antibody and then I can reprobe without any signal from the first antibody.

the milk with your secondary may have gone bad and may require replacing.

I store my primary antibodies after use at -20°C and reuse them 5-8 times! At -20°C you won't have any problems with bacterial growth. Works perfectly.

rkay447 on Mar 24 2009, 12:54 PM said:

NaAz is quite toxic. How do you handle your antibody, your membrane and everything that was in contact with it?
Cause when I'm working with NaAz, I dispose everything as hazardous waste.

We reuse our antibody solutions for months unless bacterial growth occurs. The trick is to not use milk. By diluting our antibodies in TBST rather than milk and storing at 4°C, they last for months. Comparative blots run months apart show similar staining for all of our antibodies. Non-specific binding even appears to decrease over time slightly.

Dr Teeth on Mar 26 2009, 05:01 AM said:

Just wondering, Maybe if you filtered before storage,i.e. its sterile. it should last longer right even with milk

MaggieRoara on Mar 30 2009, 05:37 AM said:

even properly handled and stored, milk doesn't last long (unless it is frozen).

mdfenko on Mar 30 2009, 11:11 AM said:

When you do this, how many times do you usually freeze and thaw before its becomes non-viable?

MaggieRoara on Mar 31 2009, 09:40 PM said:

we don't use milk for that reason. we use bsa and/or serum when we prepare our antibody solutions. even so, the proteins can (and do) go "bad" over time (just smell the solution, you'll know when).

as for how many freeze/thaw cycles the solution can withstand, you may have to determine that for your antibody preparation.