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Assay in Blood - sticky protein (Mar/14/2011 )

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Another option...lyse the blood with water prior to analysis. the spike and recovery should indicate if the analyte sticks...

-PAO_ahac-

Andrea Mica on Mon Mar 14 16:03:41 2011 said:


K.B. on Mon Mar 14 12:17:25 2011 said:


1. Do you have standard of that protein and does the assay work with that standard?
2. Are your antibodies suitable for the assay you use? (eg. Ab's for WB may not be suitable for ELISA etc.)


1.Thanks, yes we have standard protein and our assay works well with the protein in buffer or urine.
2. The antibodies worked in ELISA with buffer or urine, but looking in serum we get too much noise too little signal.


re: 2. I was thinking about western blotting, not whole blood. Some antibodies for W-blot are not good for ELISA, but it's not the case if you can detect the standard.

Is it possible that this protein is being cleaved? Perhaps you should think about adding some protease inhibitor immediately after blood is collected or serum separated? Or maybe those are only some more or less specific interactions between proteins? In this case you may try adding a little bit of detergent eg. Tween-20.

PAO_ahac is right - you should try spiking serum with standard.

-K.B.-

re: 2. I was thinking about western blotting, not whole blood. Some antibodies for W-blot are not good for ELISA, but it's not the case if you can detect the standard.

Is it possible that this protein is being cleaved? Perhaps you should think about adding some protease inhibitor immediately after blood is collected or serum separated? Or maybe those are only some more or less specific interactions between proteins? In this case you may try adding a little bit of detergent eg. Tween-20.

PAO_ahac is right - you should try spiking serum with standard.

Thanks for the answers, there are a few things for us to try.

-Andrea Mica-

PAO_ahac on Thu Mar 17 10:35:07 2011 said:


Another option...lyse the blood with water prior to analysis. the spike and recovery should indicate if the analyte sticks...

Thanks PAO-ahac,

I think we'll try a few of the suggestions and see what sticks (or doesnt)

Andrea

-Andrea Mica-
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