Question (50595) | GST 6XHis-tag ELISA Kit (ab128573)

Go to datasheet (ab128573)

Question

I created a virus that produces protein with a 6xHis at the end - the protein is soluble and indeed it is detectable in serum. I have tested it cell culture with success and need to test it in serum.

Answer

Thank you for your reply.

The lab has sent me the following information and advice:

This kit can be used to measure recombinant/expressed proteins tagged with 6xHis. Two examples are shown in the datasheet: GSK3B-his tagged and GST-his tagged.



The kit does work on serum (see image at the bottom of datasheet with 75% recovery rate). However, since the serum is from mouse samples, there will be too much background. What we have seen in the literature, is that researchers use these type of kits with serum samples to detect humoral response against His-tag. In this case, the researcher adds the serum as a detector antibody and not as a sample. In this case the sample would be the recombinant protein provided. But if they want to use the serum as a sample it won’t work due to mouse on mouse issues.



One potential idea could beto first deplete the serum sample from IgG molecules by incubating for a couple of hours on a GAM coated plate. Then it could be transferred to the His Tag plate. Although, it might be difficult to remove all IgG completely, it would most likely be only a depletion. However, if combined with a WB, this might work.

Another method would beNickel coated plates (which we unfortunately don't carry). This will probably work a lot better for you (just one step without having to deplete the sample of IgG).

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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