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Diluting antibody stocks in glycerol


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8 replies to this topic

#1 science noob

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Posted 20 March 2012 - 06:53 PM

Would this affect the final staining/visualisation?

#2 doxorubicin

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Posted 21 March 2012 - 10:58 AM

No. It is common practice to supply antibodies in 50% glycerol so that they won't freeze at -20C.

#3 bob1

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Posted 21 March 2012 - 11:51 AM

Using glycerol as the diluent would be silly, it is very viscous and hard to measure out, so your dilutions would be inaccurate, and incubating in glycerol may (should) change the diffusion rate of the antibodies - they would move much more slowly, so you would likely have to incubate for much longer.

#4 doxorubicin

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Posted 22 March 2012 - 01:31 AM

I believe the original question was whether the antibody stock being diluted in glycerol would pose any problem. Since this antibody stock would likely be diluted at least 1:100 for staining purposes, there would not be any possibility of slow moving antibodies (is this even a thing?). As for the pipetting, it is very easy to pipette 20ul of a 50% glycerol solution. I agree a 100% glycerol solution might be difficult to pipette accurately.

#5 bob1

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Posted 22 March 2012 - 11:35 AM

I (obviously) parsed that as diluting the stock in glycerol, not that the stock was already in glycerol and further diluted. I agree a 50% glycerol solution is relatively easy to pipette.

Diffusion rate is definitely a phenomenon...

#6 science noob

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Posted 22 March 2012 - 02:30 PM

So, what is the consensus? I see two schools of thoughts on this.

I will try to make sure to make a correct dilution (1:1 glycerol-antibody). Ultimately, the staining is what I'm worried about since glycerol could interfere with the visualisation (or not)?

And yes, the main reason for doing this is so that antibody stocks doesn't freeze at -20 C since we will be freeze-thawing it regularly. I've also aliquoted them into aliquots.

#7 doxorubicin

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Posted 23 March 2012 - 12:01 AM

Some companies (e.g. Cell Signaling Technology) supply all of their antibodies in 50% glycerol, and these antibodies have been used in thousands of publications for staining purposes. Your question seems to be whether the results will be the same if you dilute your stock solution 1:2 in glycerol for storage purposes, prior to diluting (say 1:100) in your normal staining buffer. So your question is whether the presence of 0.5% glycerol in your primary antibody staining step will change the result. I say "no", but I guess there is only one way to find out. Maybe bob1 has some estimates on the relative diffusion rate of antibodies in normal staining buffer vs. staining buffer with 0.5% glycerol and has calcuated how fast the orbital shaker has to be spinning to overcome this difference (sorry couldn't resist).

#8 leelee

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Posted 23 March 2012 - 12:57 AM

From the Abcam website:
"Some researchers add the cryoprotectant glycerol to a final concentration of 50% to prevent freeze/thaw damage; glycerol will lower the freezing point to below -20oC. While this may be acceptable for many antibodies, only a small percentage of the antibodies we offer have been tested for stability in this storage condition and our guarantee only applies to antibodies stored as recommended on the data sheet."

Which would make me think that it would might be good idea to check if using glycerol will have an effect on your staining empirically. I don't see why they would make such a claim if it hasn't been a problem for someone along the way.
Although as doxorubicin states, many antibodies do come in buffers containing glycerol, so the likelihood of any adverse effect is probably low.

(bob1, I too read and understood the original post as you did..... )

#9 mdfenko

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Posted 23 March 2012 - 09:55 AM

glycerol by itself should have no effect on staining.

you have to remember, though, that the antibody concentration will now be half that of the original. instead of using a 1:100 dilution you will now need to use a 1:50 dilution of the glycerol stock to get the same concentration of antibody in your working solution.
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