Question (74236) | Antibody Concentration Kit (ab102778)

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Question

I will try to conjugate the anti-4 Hydroxynonenal antibody (ab48506) to FITC by using EasyLink FITC Conjugation Kit, according to your suggestion. I read the antibody concentration kit datasheet and I was wondering if better instruction can be provided about the Buffer Exchange protocol. For instance, is there a spin speed that is ideal (an optimum spin speed) and how many minutes are required to reduce the buffer volume from 500 to 100 microliters?

Answer

Most benchtop microcentrifuges have a maximum spin of 14,000 - 15,000 RPM, and a maximum RCF of 18,000 - 21,000 x g. Anywhere in that range will be fine. You might want to test spinning just buffer without antibody, to see how long it takes to reduce 500 ul (or less) to 100 ul, but an antibody solution will likely take longer, since the antibody blocks the filter. You can always check every 30 seconds to see how much the volume has been reduced, taking notes. After the first spin, the subsequent spins should take the same amount of time, assuming you add conjugation buffer after each spin (for example, PBS) to bring the volume up to the original volume of the Tris-buffered antibody solution.
The hydroxynonenal antibody ab48506 will be 50 ug in 500ul, a concentration of 0.1 mg/ml. You will want to concentrate the antibody with the same spin column by not adding more buffer after the last buffer exchange spin. Ideally, you want a concentration of at least 1.0 mg/ml for efficient conjugation.

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