How to reuse antibody in Western blotting - Share your experience (Jul/04/2008 )

I had reused my antibody for like 3 times. I used the antiphosphate histone antibody from Cell Signaling. I kept in -20C.Apparently, all I get in the end is just black smearing. I think I should throw the antibody away. That is quick isnt it? I thought I can reuse it like 5 times at least.

Is it normal? Thanks!

---

I assume you are talking western, and that what you are reusing is the antibody diluted in whatever the buffer you use for blotting (PBS etct). If storing at -20C does this means that every time you've reused it you have thawed and freeze it again ? In my experience this might hugely affect the stability of the antibody and you might be better to store at 4C between uses.

---

Did you add azide? Quite necesary for re-use even if storing at -20 in-between.

---

I would say it depends on the antibody you are using. Some antibody are not reusable, but others are for up to 10-15 times. As for the smears, it may be due to protein degradation, or lack of blocage. Reusing antibody means reusing the same solution to block. So you may try to re-add milk or bsa (whatever you block with).


And as said almost a doctor, freeze/thaw is not good for an antibody, but once again, it depends on the antibody itself.

---

Several previous discussions on this topic:

How many times do you reuse a primary antibody?
reuse the primary antibody , and how many time
Reuse antibodies for further incubation in Western blot

---

you may reuse within one week, storing diluted Ab at 0-4°C; longer storing at 4°C, denaturizing of Ab is observed (I tried to stabilize with BSA but it accelerated falling out); freezing without freeze buffer may destroy Ab;

the question is how to stabilize a diluted Ab solution to store at 4°C for long term; NaN3 is a preservative but does not prevent denaturizing

---

hi,
as members mentioned add 0.1% NaN3 as preservative.
add 0.05% or 0.1% gelatin. this will keep your antibody stable for long time. if you are not sure just give a try ;-)

gud luk
sravan

---

Reusing is really dependant on the kind of antibody, how good does it work at first time? If you get great signal with strong intensity, it is not so bad if you lose a bit of activity by freezing, even at -20°C. But if it is a very weak and unstable one, you will destroy it and get nothing the 2nd time. Be careful when you add NaN3 to it if you are using ECL, this impairs the peroxidase function, so you have to wash it away before 2nd antibody really well.
But storing at 4°C results in fast bacterial or fungi growth, if you have blocking reagent like BSA or milk in the buffer after a few days.

This is not wholly true since you block 1h before with fresh blocking solution. This will do most of the effect and the incubation of the first antibody in buffer with blocking reagents just adds to the effect. You can also but blocking reagent in the second one (if you are not already doing this) and the effect will be intensified.

---

good, thank you too! Touche!

---