Antibody Purification Kit (Protein A) (ab102784)

As we discussed, our studies have found little effect on the conjugation chemistry with up to 0.05% BSA and up to 0.1% glycine in the antibody storage buffer. Thus, I think you do not have to worry about those components. However, as you know the 0.01% sodium azide will inhibit HRP activity, and as you mentioned the 50% glycerol is making it hard to remove the sodium azide.

Antibody purification kit if you are going to stay with ab76424:
Protein A for rabbit antibodies ab102784: https://www.abcam.com/Antibody-Purification-Kit-Protein-A-ab102784.html
Protein G for other species ab 128747: https://www.abcam.com/Antibody-Purification-Kit-Protein-G-ab128747.html
For mouse antibodies ab128745: https://www.abcam.com/Mouse-Antibody-Purification-Kit-ab128745.html

Previous customers have contacted us with issues purifying antibodies with high concentrations of glycerol, and we found the following solution proved successful:
" When using the antibody purification kit the presence of glycerol in the antibody buffer will make it more viscous. This means that it will require more G force to pass through the spin column. It is possible to buffer exchange the antibody it just means the customer will have to spin for much longer. Alternatively, a simpler solution would be to dilute the antibody. This would reduce the glycerol’s concentration, which in turn will make the buffer less viscous and easier to spin out. You can then use the same spin column to concentrate the antibody back to its original concentration and volume, after having performed the buffer exchange."

Additionally, we have our EasyLink conjugation kits, including HRP conjugation, listed through the link below. Our EasyLink conjugation kits are capable of fully successful conjugation with glycerol concentrations of up to 50%. We are currently running a promotion for an extra 20% off our EasyLink kits:
EasyLink Kits: https://www.abcam.com/index.html?pageconfig=resource&rid=13148#enzymatic
Easylink Promotion:https://www.abcam.com/index.html?pageconfig=resource&rid=15389&source=pagetrap&viapagetrap=easylink123
Below is a list of compatible and incompatible buffers with our EasyLink conjugation reactions:
https://www.abcam.com/index.html?pageconfig=resource&rid=13156#11

Thank you for your call and your patience while I communicated with my colleague in the lab.

In response to your inquiry, it turns out that both options (ab102784 Antibody Purification Kit (Protein A) or ab102778 Antibody Concentration Kit ) will work for your antibody purification purposes. However it turns out that the simplest kits would be the Concentration kit.

The spin columns provided in this kit allow a simple buffer exchange by ‘spinning away’ the existing buffer, and allowing this to be replaced by a more compatible buffer. As spinning the antibody dry is not best practice, as this can impact the antibody, the protocol states that this allows the azide to be reduced, as there will be a small amount of azide left in the little bit of buffer that remains. However if this process is repeated, each time, the amount of azide remaining will reduce more and more, therefore after enough spins, the azide concentration will effectively become zero. On the whole, this azide should be reduced to a suitable level almost after 1 buffer exchange, however we would recommend this is repeated further if the label of choice is HRP, as this has shown to be more sensitive. I hope you find this information helpful.

Please let me know if I can offer any more help or technical advice. Good luck with your research.

In cases where the antibody volume is larger, i.e. in your case where you have 1ml of antibody, we recommend you do the following:



First, add the binding buffer to your antibody. Then add the binding buffer and antibody mix to the Protein A resin. You might want to add a small amount initially to reconstitute the Protein A resin in its original vial, then move the reconstituted resin along with the remaining antibody/binding buffer mix to a separate tube - preferably a 2ml (or bigger) eppendorf tube - as the one we provide the resin in might not be large enough to hold the entire volume.



Then incubate the antibody/binding buffer/Protein A resin mix for 1 hour.



After the incubation, add your antibody mix to the spin cartridge and spin down. You should spin down the entire antibody mix in aliquots, as the entire volume will not fit in the spin cartridge.



After spinning down all your antibody, continue as normal with the protocol by adding the wash resin to the spin cartridge.

Protein values should be determined using an absorbance at 280nm. I have checked with our scientists, and they informed me that an absorbance (or OD) of 1 at 280nm corresponds to an antibody concentration of 0.1714mg/ml. Please note this value is true only for IgG’s measured in a 1cm cuvette.
You should be able to find the appropriate absorbance-concentration coefficient for the antibody you are trying to purify by looking at references and other sources, which can be readily found online.
According to http://www.mainebiotechnology.com/documents/3528_C007.pdf The following protocol can be used:
ANTIBODY CONCENTRATION BY ABSORBANCE AT 280 NM
One of the fastest methods for determining purified antibody concentration is by absorption at 280 nm (A280). For this, an ultraviolet-visible spectrophotometer is needed to detect light transmitted in the ultraviolet range. Proteins that contain the aromatic amino acid residues tryptophan, phenylalanine, and tyrosine absorb light energy at a wavelength of about 280 nm. Each protein has its own extinction coefficient based on the known number of these three amino acids. Here, in the case of immunoglobulins, the compositional differences can largely be ignored. The greater the absorbance, the higher the concentration of protein. To obtain satisfactory results, the preparation must be >95% pure.
Procedure
1. Turn on spectrophotomer and adjust setting to 280 nm.
2. Dilute sample so that the protein concentration is estimated to be 0.5–1.5 mg/ml. If the sample is highly concentrated, prepare two different dilutions (e.g., 1:5 and 1:10) to confirm results.
3. Fill a cuvette with diluent. When the spectrophotometer has had ample time to warm up, insert the cuvette with the diluent and adjust the reading to “0.”
4. Empty the cuvette and tamp dry. Transfer enough of the more diluted sample to the cuvette, mix briefly and discard this sample. Transfer another volume of the same sample to the cuvette. Insert into the spectrophotometer and record the value.
5. Discard the sample. If there is a second, more-concentrated sample, transfer a volume of that to the cuvette. Again, mix briefly and discard. Transfer a volume of the same sample and insert into the spectrophotomer. Record the value.
6. Determine the concentration of the diluted sample. The absorbance reading of the sample is divided by the proper value displayed in Table 7.2. The chart indicates what the absorbance reading should be (extinction coefficient) if the antibody concentration is at 1.0 mg/ml.
7. Thus if an IgG sample absorbance is 0.75, then the concentration is 0.75 ÷ 1.36 = 0.55 mg/ml. If this happens to be a 1:5 dilution of the original sample, then the concentration of the original sample is 0.55 × 5 = 2.75 mg/ml.
Extinction Coefficients of Various Immunoglobulins
Protein Moiety Absorbance at 1.0 mg/ml
IgG 1.36
IgM 1.18
IgA 1.32
IgE 1.53
IgG F(ab) 1.50
IgG F(ab)′2 1.48

Thank you for contacting us.

I am afraid that this kit doesn’t work for purification of IgMs. IgMs are normally purified by precipitation.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

This kit will certainly suit your needs for removing the Tris-glycine and glycerol from your antibody before use with the EasyLink conjugation kit. While the EasyLink kits are able to be used with as much as 50% glycerol content they do need Tris levels which are below 20mM. When using the antibody purification kit the presence of glycerol in the antibody buffer will make it more viscous. This means that it will require more G force to pass through the spin column. It is possible to buffer exchange the antibody it just means the customer will have to spin for much longer.  Alternatively, a simpler solution would be to dilute the antibody. This would reduce the glycerol’s concentration, which in turn will make the buffer less viscous and easier to spin out. You can then use the same spin column to concentrate the antibody back to its original concentration and volume, after having performed the buffer exchange. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Thank you for your enquiry.

We would recommend to aim for a maximum of 15,000g. Lower speeds will also be fine, it just may require slightly longer spin times to achieve the objective.

I hope this will help. If you require any further information, please let me know.

Thank you once again for your reply and patience.

I am pleased to now have further information for you. We would recommend to use the following steps in this case:

Add the resin to the 1 ml of antibody in a 1.5 ml eppendorf. Then add the binding buffer and do the incubation. One point to note is that as the volume has been increased, it would be advisable to increase the incubation time with the resin. Double the volume equals double the incubation time.

After the incubation step, spin the eppendorf in a microfuge (at max speed for 3 mins) and remove the supernatant. It is then best to keep the supernatant safe until a successful purification.

Finally, re-suspend the resin in 0.5 ml of wash buffer.

Transfer to the spin column and follow the protocol as normal.

If you have any additional questions, please do not hesitate to contact me.

Thank you for contacting Abcam.

Please find the link to the antibody purification kit below, that will allow you to remove the sodium azide from your antibody solution:

https://www.abcam.com/antibody-purification-kit-protein-a-ab102784.html



If there is anything else I can help you with, just let me know.

Thank you for bringing this to our attention. Assuming you followed the incubation and elution protocol, I am not sure what could have gone wrong. We have not had any complaints in the past several months. Did you try getting an absorbance reading for the washes, or test a solution of BSA, for instance 0.25 mg/ml? Did the column dry out at any point?