Question (43465) | Anti-Geminin antibody (ab104306)

Go to datasheet (ab104306)

Question

Dear xxxx,

Thank you very much for making the proper arrangements for the antibody to be sent. I'll let you know about the results I get as soon as possible.

I also appreciate the information provided regarding with the ab104306, ab102778, ab81032 and ab102914. I'm more than happy to place the order today.
I found the FAQs very helpful, however, I just have a question that I still don't know how I'm going to approach -experimentally wise- as the protocol isn't completely clear for me (It might be just a language barrier but I think it is worthwhile to ask).

When concentrating the antibody...
After placing the antibody within the spin cartdrige I have to do the following:
"Spin for 1 to 3 minutes in a microfuge at max speed of 15000g to reduce the buffer volume in the spin cartridge to between 50 and 100ul. It advisable not spin the antibody dry as reconstitution of the antibody will be difficult and significant antibody loss and degradation may occur".

My question is the following:

Does the bold text mean that -for example-, if I put 100uL of my antibody in the spin cartdrige then
a) A volume of approx 50 to 100 uL will be removed out form the cartdrige ? or
b) That the remaining volume in which the antibody will be contained after being spun down will be from 50 to 100 uL (e.g 75 uL). This step isn't clear enough for me I'm afraid and I wouldn't like to spin dry my antibody!!

Thank you in advance,

Looking forward to hearing from you,

Answer

I am sorry that the protocol details were not explained clearly enough. I will try to explain as best I can, if it does not make sense please let me know and I'll try to rephrase.

You would take your antibody (concentration of 0.36 mg/mL), you are hoping to achieve a concentration of at least 1 mg/mL to use it with theconjugation kit. In order to do this, you could concentrate all the antibody you will be provided with:

100 ug of antibody at 0.36 mg/mL, gives you 277.7 uL in total. This therefore needs to be concentrated by about a factor of 3 to get the desired concentration. You would therefore load the antibody into the spin cartridge. Spin (start with no more than 1 minute at no more than 15,000g), this will give you an idea of how quickly the solution will be removed. Continue to spin (checking after every minute or less or centrifugation) until you have removed ˜185 uL of solution. You should therefor be left with ˜92 uL of antibody at ˜ 1mg/mL.

If you would like to concentrate only a portion of the antibody, the same process can be followed but reducing the volume added and the volume discarded. Be very careful however to not let the antibody come to complete dryness (try not to reduce the antibody volume to below50-100 uL).

I hope this has helped to clarify the procedure. If you have any further questions, please do not hesitate to contact me again.