Can anyone recommend a method or product to remove glycerol from small volumes of affinity purified antibody?
We are using Dynabead M270 Epoxy beads to couple antibodies to beads. This kit specifically discourages against using antibody containing glycerol. Unfortunately, some of our antibodies were stored in 30-40% glycerol. We would like a way to remove the glycerol and other additives from the antibodies that has both a high yield and is cost effective.
I've tried using Pierce Protein A/G beads to purify the antibody (as per manufacturer protocol). Unfortunately, when using this "purified" antibody with the Dynabead coupling, the ability of the antibody to coIP proteins was significantly reduced.
Any recommendations are welcome Thanks!
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UPDATE:
I've since developed an easy protocol to deal with glycerol removal using amicon columns. Since a few people have messaged me asking for this protocol, I've attached my response below:
The glycerol removal with the amicon columns is a total buffer exchange. The Amicon 10K columns keep any proteins above 10K kD attached to the membrane, allowing the buffer to flow out. You then resuspend the antibody in a buffer of your choice (I use PBS).
I've listed my protocol below. I would recommend testing the antibody +/- the glycerol cleanup, and make sure there is an improvement. I've found that some antibodies perform better without the cleanup, even if there is glycerol in the buffer.
http://www.emdmillipore.com/US/en/product/Amicon-Ultra-0.5nbspmL-Centrifugal-Filters-for-DNA-and-Protein-Purification-and-Concentration,MM_NF-C82301
Using the: Amicon 10K Buffer Exchange Column (EMD, Cat # UFC501096) 1. Bring antibody to 500 ul with sterile PBS. 2. Place Amicon 10K spin column into supplied collection tube 3. Add 500 μl of antibody in PBS to spin column. Close lid. 4. Spin for 20 mins at 14K, at 4C a. For all spins, place elbow of tube inwards 5. Discard flow through 6. Add 500 μl PBS to spin column 7. Spin for 15 min at 14K, at 4C. Then proceed immediately to next step 8. In new collection tube, place spin column upside down 9. Spin for 2 min at 1K, at 4C to collect buffer-exchanged antibody Save eluted antibody and add 50 μl - 220 μl of PBS (solution will be slightly viscous). Store on ice until needed.
Thanks for the useful pointers everyone. I ended up removing the glycerol with a amicon spin column (essentially a buffer exchange). This worked well and improved IP efficiency with the antibody.
I too was still confused about the glycerol effect and wrote to Dynabead's tech support. I received the following explanation as to why glycerol is detrimental:
"it seems that the glycerol will not damage the protein as implied in the handbook, but it will interfere with the coupling chemistry by reacting with the epoxy groups at a low level. Therefore it would be best to remove it. As glycerol has 3 OH groups this may be what caused the beads to aggregate"
Hi,
Have you though of dialysing your sample? I have used devices such as Float-A-Lyzer to remove unwanted compounds in the buffer, and in some cases also concentrated the sample by simply placing the tube on solid PEG that leeches water out of the tube.
Hi,
Dialysis should work fine. However it is often forgotten that it is a very slow process. I would suggest using ca 50 volumes of PBS for 36 hours. During this time replace the buffer 3 times.
Your antibody solution will increase in volume during dialysis so remember to use extra tubing
Hope this helps
In the protocol for Dynabeads M-270 Epoxy, I could not find any comment on glycerol. In addition, I would not expect a detrimental effect of glycerol on your conjugation, except the viscosity, which can be easily reduced by dilution in a suitable buffer, if the concentration of your antibody stock solution is high enough.
https://www.thermofisher.com/order/catalog/product/14301
Interestingly, the "Dynabeads® Antibody Coupling Kit" (cat 14311D) which also uses M270 epoxy, clearly states in the manual that;
Coupling of antibodies or other proteins stabilized in glycerol is not recommended. Although it is possible to couple such ligands, the antibody (or protein) function may be severely negatively affected.
https://tools.thermofisher.com/content/sfs/manuals/dynabeads_antibody_couplingkit_man.pdf
From my point of view, the statement in this manual is strange. Glycerol is one of the best stabilizers for proteins, including antibodies. Can anybody give a rationale for the comment in the protocol?
Thanks for the useful pointers everyone. I ended up removing the glycerol with a amicon spin column (essentially a buffer exchange). This worked well and improved IP efficiency with the antibody.
I too was still confused about the glycerol effect and wrote to Dynabead's tech support. I received the following explanation as to why glycerol is detrimental:
"it seems that the glycerol will not damage the protein as implied in the handbook, but it will interfere with the coupling chemistry by reacting with the epoxy groups at a low level. Therefore it would be best to remove it. As glycerol has 3 OH groups this may be what caused the beads to aggregate"
To remove low molecular weight compounds from high molecular weight compounds from aqueous solution previously scientist were either using dialysis or gel chromatography. It was depend on the amount of high molecular weight compound present in the aqueous solution. The above procedure are redundant due to availability of spin columns for the separation of low molecular weight compounds from high molecular weight compounds present in aqueous solution. Because this procedure concentrate samples, one can wash the samples with requisite buffer required for storing or coupling and takes less time.
With best wishes.
"...but it will interfere with the coupling chemistry by reacting with the epoxy groups at a low level. Therefore it would be best to remove it. As glycerol has 3 OH groups this may be what caused the beads to aggregate"
As a chemist, I consider this kind of reaction as highly unlikely in an aqueous environment under these conditions.
Hi Michael -- interesting insight. As a chemist can you hypothesize what the issue might be? I've attached the Thermo's description of the Epoxy m270 chemistry.
Hi Samantha, as you can see in the description of Thermo, only amines or sulfhydryl groups are reactive enough for epoxides under normal conditions (not hydroxyl groups as in glycerol). It is true that high-salt improves the immobilization of many proteins. However, most amines are protonated at neutral pH, which makes them quite slow. You may use high pH (critical for proteins), high temperature (also bad for most proteins) or a long reaction time (12-48 hrs). As I mentioned already, the viscosity of the glycerol might be the only obvious drawback. This makes diffusion slower and hence reduces the reaction yield. However, this effect can be easily reduced by dilution with incubation buffer, which is often performed anyway or by increasing the reaction time.
By the way: Antibodies might be damaged by impurities of glycerol if glycerol of insufficient quality has been used. Unfortunately, this damage is irreversible and can not be changed by removal of the glycerol.
I've had a few people ask for recommendations on my glycerol removal step. I've added my protocol as an update to the question. Hope this helps others!
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