Antibody titration

Hello everyone. I have started doing IPs and Western's following other people's protocols.

Can anyone give me an easy way of doing antibody titrations for blotting. Im not the best at math.

I need to titrate a primary antibody 1:1000 and its [conc] is .75 mg/ml

in 7.5ml 1x blocking buffer and 7.5ml 1x TBST so its final volume is ~15ml

and likewise and HRP conjugated Goat anti-Rabiit at .4mg/ml

which is titated 1:5000. Thanks.

Also, I have been told in the past that secondary Abs can be diluted down to even 1:10,000 because they bind so well. Is this true? What are other experiences?

I do not see what you mean "in 7.5ml 1x blocking buffer and 7.5ml 1x TBST so its final volume is ~15ml", by my understanding, you probably mean 7.5ml 1X blocking buffer in 1X TBST, that is, you make your blocking buffer with TBST

Since you have your primary antibody at .75 mg/ml (very concentrated), I will suggest start with 1/3000~5000, instead of 1/1000. Which is, you will need 1ul of your antibody for every 3~5ml of your solution

For 2nd antibody, start with 1/10000 would be wise. which is, you will need 1ul for every 10ml solution

If your result happens to be very dark (seems to me it will, if your antibody is.75 mg/ml), then try to dilute your 1st antibody to 1/10000, etc.

good luck

I am not sure if I understand your question. You have 15 ml of a 0.75 mg/ml solution and you want to make a 1:1000 dilution? If your goal is to titrate your antibody all you need to do is dilute your antibody (I would start with 1:500, 1:1000 and 1:2000 - to make sure you get a signal) to compare it against your secondary antibody in your protein samples. I assume your goal is to make sure your primary antibody+secondary gives you a high signal relative to any background the secondary alone generates.

As for your secondary, a 1:10,000 dilution will work, but this depends on how good the secondary is. What I recommend is to stick to the 1:5,000 dilution and simply reuse it 2 or 3 times. You do not want to have limiting secondary antibody.

PS. I agree 0.75 mg/ml is concentrated, but in the end it boils down to how good your antibody is.

Thank you for all your help. I meant that I am adding the antibody to a TBST and Blocking buffer mixture of total 15ml. I have tried 1:1000 and it gets very dark like, I can see some very dark heavy and light chains. But, I would critize reusing secondary antibody. I have always been advised not to preserve and use it more than once pretty much

a.) because its cheap

b.) because you get less HRP signal.

I understand your concern about re-using secondary antibodies. If you are concerned about carry-over then do not do it. And I do agree that you get lower HRP signal if you reuse the secondary (most of my experience reusing secondaries was with AP-labeled secondaries).

"I have tried 1:1000 and it gets very dark"

So, I would suggest go directly above 1/5000, seems to me 1/10000 could be good.

Let us know what happened next.

I wasn't aware that you were actually able to re-use secondary antibodies.  I did try it once and my blot didn't work so from then on (along with my primaries), I always used a fresh antibody stock

Yes, I too found out the hard way. Reusing secondary that is HRP conjugated gives you nothing.

Also I tried the 1:1000 dilution which yielded very dark bands. I am going to take my 15ml solution which is sitting in thimersol preserative and dilute that down by a factor of 5.

Does anyone have experience in reusing primary Ab solution of multiple blots. Can you probe more than 2 times before the signal really starts to wane?

Sure you can re-use you 1st antibody multi-times, I usually reuse mine for 3-4 times (just mark down the date and time used after every usage and keep -20, then decide whether or not to re-use it  on the most current result).

If you decide to do 1/5000 next time, just dilute your 1/1000 5 times.

and I will never ever re-use 2nd antibody, and will not suggest

Just an update. Yes the 1:3000 primary Ab dilution still produced smeering. But, I have suspicion that I need to dilute my secondary Ab. People have told me in the past that you don't need much an even 1:10,000 can be too much. So that's what I'm working on optomizing. My bands came up, just trying to get rid of residual noise.

I have also found that when incubating with Ab, blocking, etc. Using an orbital shaker versus one that rocks back and forth produces a lot less background noise in the middle of one's gel.

You may also want to decrease your protein loading, according to what you have, I will suggest half loading + 1/10000 primary antibody, I will also stick to the 1/10000 secondary antibody.

I routinely use 1:25000 for anti-rabbit secondary (Bio-Rad).  even had to drop down to 1:50,000 a couple of times.  Good luck

I also use a 1:20,000 dilution for some secondary antibodies, including BioRad goat anti-rabbit.

When I am planning on reusing the primary antibody I add sodium azide to it. It is important to wash this off well though because it can inhibit the HRP reaction. I have never reused the secondary antibody. To save antibody I often put the blot in a 50 ml conical tube (wrapped around the inside) on a rotator and then about 6-7.5 ml of antibody solution is plenty.