Question (67009) | Streptavidin Conjugation Kit (ab102921)

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Question

I came across your Easylink Biotin conjugation kit online and I would like to know if you have a way of determining your MSR after biotinylation. I would like to know the ratio of biotin per antibodies. Is this kit able to reach biotinylation levels between 6-8 biotins per antibodies?
I also would like to know if your Strepavidin Conjugation kit to conjugate antibodies provides a way to determine the ratios of strepavidin per antibodies. Is it a robust process that would conjugate all antibodies (polyclonals and monoclonals) in a consistent way?

Answer

Thank you for contacting us.

With regard to the number of biotins, each antibody will be conjugated with multiple biotins, getting up to 6 or 8 biotins per antibody is feasible. It is important to remember that you only need one biotin attached to enable the biotinylated antibody to bind to streptavidin. There are assays available to measure biotinylation. However, in this case they will not be suitable as there is no separation step post conjugation.

With regard to Streptavidin, you can only confirm the streptavidin is attached to the antibody by size. The easiest way is to run an ID gel and measure the molecular weight. An antibody is 160KD and streptavidin is 53KD. A conjugate will therefore by 160 + 53KD (one strep) or 160 + 106 (two streps) or 160 + 159KD (3 streps). In reality you will get a mixture of all three. Also you must run a non-reducing gel otherwise you will get a mess due to all the possible combinations as both proteins are composed of subunits.

You can control the molar ratio by adding different amounts of antibody. This will guide the actual labelling to a desired ratio. For example, adding the following amounts of antibody will give different molar ratios:

100ug of antibody to 100ug of strep = 3 streps on each antibody

150ug of antibody to 100ug of strep = 2 streps on each antibody

300ug of antibody to 100ug of strep = 1 strep on each antibody

This is only a guide and it will not be absolute, as no reaction is 100% efficient.

As for the question “Is it a robust process that would conjugate all antibodies (polyclonals and monoclonals) in a consistent way?” If the same antibody, either monoclonal or polyclonal, is used in the same conditions (buffers, incubations times, etc) the results will be very reproducible. If labeling is carried out with different antibodies then there might some variation between results.

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

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