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Qubit Protein Quantitation Issues


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#1 louhazosc

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Posted 31 October 2009 - 02:05 PM

I currently have a demo of an Invitrogen Qubit with the Quant-it protein kit and their specific 500uL tubes.
Of course I have to try and sell this to my PI so I am hitting it with everything I can by performing samples in triplicate, at 3 different dilutions, as well as a bradford and BCA of the same sample for comparison.

My biggest issue thus far is checking varying dilutions. I'll do this by adding 5uL, 10uL, and 20uL to their master mix of 195uL, 190uL, and 180uL respectively. I get my reading and plug that into the equation of [concentration = reading given in ug/mL x (200/volume of sample)], pretty simple. Yet I get 3 totally different values. I mean really different such as 100ug/mL, 250ug/mL, and 400ug/mL.

I think I am, capable of carrying out that equation so is there something I'm missing or have others seen this discrepancy as well.
This thing gets pretty good reviews and would love to use it over pipetting out BCAs and Bradfords.

I apologize that I do not have my notes handy and can not say which concentration goes with which volume of sample added.

#2 mdfenko

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Posted 03 November 2009 - 08:04 AM

looking at the manual brings up a couple of things...

first, are there any detergents in your samples' buffers? they are not recommended for use with the quant-it protein assay.

second, do you add the samples' buffers to the standards? it is recommended with buffer components that may have some effect on the assay (see the appendix in the manual).

finally, the standard curve is not truly linear (as shown in the manual). a simple linear calculation will not give correct results, you need to fit the curve and compare the sample to that.
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#3 PhD Scientist

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Posted 03 May 2011 - 06:49 AM

I currently have a demo of an Invitrogen Qubit with the Quant-it protein kit and their specific 500uL tubes.
Of course I have to try and sell this to my PI so I am hitting it with everything I can by performing samples in triplicate, at 3 different dilutions, as well as a bradford and BCA of the same sample for comparison.

My biggest issue thus far is checking varying dilutions. I'll do this by adding 5uL, 10uL, and 20uL to their master mix of 195uL, 190uL, and 180uL respectively. I get my reading and plug that into the equation of [concentration = reading given in ug/mL x (200/volume of sample)], pretty simple. Yet I get 3 totally different values. I mean really different such as 100ug/mL, 250ug/mL, and 400ug/mL.

I think I am, capable of carrying out that equation so is there something I'm missing or have others seen this discrepancy as well.
This thing gets pretty good reviews and would love to use it over pipetting out BCAs and Bradfords.

I apologize that I do not have my notes handy and can not say which concentration goes with which volume of sample added.



Same problem for me :-(:- Are there any lysis buffers that are compatible with Qubit!!!




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