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Need to find a method of detection for free Histag

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#1 paperiswhite



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Posted 15 January 2011 - 11:12 PM

So, Ive synthesized with SPPS my peptide with is just 6 Histidines (basically a free histag) and now I need a way to detect the concentration of the peptide to establish a calibration curve (I need this so I can extrapolate the concentration of any unknown sample). Ive tried a Biorad assay for protein, but the peptide would not follow a trend as I increased its concentration. I only have 20mg of the peptide so the ideal concentration range for me would be in the micromolar or nanomolar range (which I assume is way too low for the Biorad assay). A colorimetric method is preferred, because of the the simplicity of UVvis, but any method that can be rapidly performed for multiple samples is ok.
Please help!

#2 protolder



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Posted 16 January 2011 - 10:42 PM

Hola, I think that the most accurate method able to analyze lots of samples and with low consumption of sample is an ELISA, but you need specifical plates to coat with the samples, an anti His antibody, an secondary antibody and the colorimetric substrateżDTNB? I believe remember. If you have a plate reader is the best, but you could read sample by sample in an spectrophotometer, but yuo need have your 6His bounded to any surface, to quit the bacground of not bounded antibody. Revise the method in any manual or internet and search in your institution for anybody who made ELISA, He could help you. Buena suerte

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