I am looking forward to conjugate PSA antibodies (Monoclonal, Mouse, IgG1 - A67-B/E3 ) available at
https://www.thermofisher.com/antibody/product/Prostate-specific-antigen-Antibody-clone-A67-B-E3-Monoclonal/MA1-10447)
with photo-cleavable biotin NHS ester available at (http://www.ambergen.com/technology/pc_linkers.asp).
The antibodies are coming in a solution having Sodium Azide.
Is it necessary to remove sodium azide from the solution or can it be used with it as well. Secondly, if it needs to be removed, what is the best and quickest way to remove it.
It would be great if anyone would share a protocol or a much simple approach for conjugation of PSA antibodies with the above mentioned photo-cleavable biotin.
One easy way to remove the sodium azide is to use a desalting spin column. Equilibrate the resin with the coupling buffer (such as 0.1 M sodiumn bicarbonate) before putting he protein over it. Also, consider what else is in the antibody solution besides sodium azide. Is there anything else that would interfere with the coupling, such as Tris buffer (Antibodies are usually in phosphate buffer, which should not interfere with the coupling, except for being at too low a pH)? You may need to use two spin columns in sequence to remove all of the substance if there is a high concentration of it. This will result in some loss of protein, but it is a fast technique.
Another easy way to remove interfering substances is to dialyze the antibody against the coupling buffer. This will give a higher yield of protein, but takes a whole day.
A third method is diafiltration. Concentrate the antibody at least 10-fold with a centrifugal ultrafiltration device. Re-dilute with the coupling buffer. Repeat 2 more times.
Follow Dr. Shapiro's instructions if you need to remove chemicals. However, if the antibody only has Azide, no primary amines, glycerol or other chemicals, then you should be fine. Most conjugation kits can handle azide and PBS. However, you always need to read the instructions of the conjugation kit, as each kit is different in accepting chemical contaminates.
Dear Usman
I have performed this technique thus would suggest this too...however i feel above method by Shapiro is more faster.
Sodium azide can be removed from antibody solutions by gel filtration. The molecular weight of IgG is 150,000 daltons; the molecular weight of sodium azide is 65 daltons. A micro-dialysis unit with a cut off at 14,000 daltons will retain the antibody as the azide diffuses out apart gel filtration would work out.
Good luck!
It is better to remove azide. However, it is the fact, that azide does not affect the reaction. Primary and secondary amines affect the reaction. Amines may present in the form of TRIS buffer or glycine, which was used in the elution of antibody in affinity chromatography. Protocols of conjugates synthesis with NHS esters are described in many sources. For example here Hermanson "Bioconjugate techniques"
Azide will interfere with most conjugation reactions including the one you propose. You can still do conjugation reactions in the presence of azide, but they will be much less efficient. Of course the degree of interference will depend on exactly how much azide is present and how much biotin NHS molecule is added to the reaction. You will need to use more if azide is present. Or, if you can tolerate some losses of antibody, use the methods suggested above to remove the azide.
Inorganic azides such as sodium azide are very strong nucleophiles, probably one of the strongest known and they will interfere with reaction by consuming some amount of NHS ester. This AB is offered in 100 ug amount =, which is ca. 0.67x10-9 mole (FW ca. 150,000), and I presume in is in 100 uL (10-4L). If I correct concentration of AB is around 0.67x10-5 M or 0.67x10-2 mM. It is supplied in 15 mM sodium azide, which is about 4000 fold excess. Even if 100 fold excess of labeling reagent is used, there is still about 40 azide molecules (ions) per 1 molecule of NHS ester.
I did several NHS labeling reactions in the presence of sodium azide, at best I call it inconsistent. It is very simple and quick to desalt small amounts of proteins with Zeba columns, it takes 5 min an will remove up to 95% storage buffer.
Also, there is another supplier of PC Biotin-NHS Ester https://clickchemistrytools.com/product/pc-biotin-nhs-ester/ You can also find an easy to use protein labeling on the same page.
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