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claire84's Content

There have been 3 items by claire84 (Search limited from 06-August 19)


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#170818 protein biotinylation

Posted by claire84 on 16 September 2014 - 04:39 AM in Protein and Proteomics

Hi everybody,

I have some problem with the biotinylation protocol for a protein and I need your help.

I bought from Pierce a "Sulfo-NHS-LC-biotinylation kit" and I used it to incorporate the biotin into my protein (fibronectin).The kit I bought was for the biotinylation of 1-10mg of protein as weel as the protocol. 

However, I used it to target a protein ammount inferior to that reported into the protocol. Maybe for this reason I encountered some problem regarding the incorporation. After the quantification of mol of biotin per mol of protein, I determined a great ammount of biotin into the sample. 

 

Something goes wrong in the purification step?How can I check the quantity of unreacted biotin in my final sample?

 

Do you have some experience with this kind of biotinylation kit?Please find attached the file of the biotinylation protocol.

 

thank you,

 

Chiara




#166472 Km calculation

Posted by claire84 on 31 March 2014 - 01:17 AM in Biochemistry

Hello everyone!!

I'm trying to measure the km of MMP2 in the presence of a peptide substrate. I measured the increase of fluorescence at various concentrations of substrate but I encontered some problems with high substrate concentrations. High substrate concentrations cause the saturation of the instrument signal and the increase of fluorescence is no more visible. Lowering the sensibility of the instrument signal could help to solve the problem for high concentrations, but it could be  problem for the lower ones.

Do you know how to skip this problem?

 

Thanks for any help!!

 




#165436 metalloproteinase 2 handling

Posted by claire84 on 24 February 2014 - 08:17 AM in Biochemistry

Dear all,

I need your precious help!

I'm going to start some inhibition assays to test potential metalloproteinase 2 inhibitors. 

I bought  a batch of mmp2 with a concentration of 15 uM from "Enzo life sciences". For my inhibition assay I'll need an enzyme final concentration of 2 nM, so I decided to dilute the stock solution, to aliquot it and to store the aliquots at -80 °C. 

In the product data sheet they specified that procedures such as diluition of the enzyme followed by refreezing could lead to loss of activity. So I'm no more sure, that my ideas are correct and I don't know how to handle this enzyme.

 

Do you have some experience with this enzyme?

 

Please find attached the mmp2 product data sheet.

 

Every suggestions will be very appreciated!

 

thank you in advance

 

Chiara

 

 

 

 

 

 

Attached Files





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