Sircol Assay Manual - Biocolor Assays
Sircol Assay Manual - Biocolor Assays
Sircol Assay Manual - Biocolor Assays
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PROTOCOL CONTINUED FROM INSIDE FRONT COVER<br />
Washing and Draining Gently layer on 750µl ice-cold Acid-Salt Wash<br />
Reagent to the collagen-dye pellet to remove<br />
unbound dye from the surface of the pellet and the<br />
inside surface of the microcentrifuge tube.<br />
Centrifuge at 12,000 r.p.m. for 10 minutes.<br />
Drain the wash into a waste container and carefully<br />
remove any fluid from the lip of the tubes using<br />
cotton wool buds.<br />
Release and Recovery of Add 250µl Alkali Reagent to reagent blanks,<br />
Collagen Bound Dye standards and samples.<br />
Recap tubes and release the collagen bound dye<br />
into solution. A vortex mixer is suitable.<br />
When all of the bound dye has been dissolved,<br />
usually within 5 minutes, the samples are ready for<br />
measurement. The colour is light stable, but<br />
should be read within 2 to 3 hours. Keep tubes<br />
capped until ready to measure Absorbance.<br />
Measurement Transfer 200µl of each sample to individual wells of<br />
a 96 micro well plate, (keep a record map of the<br />
contents of each well; A1 to H12).<br />
Set the microplate reader to 555nm, or the closest<br />
matching blue-green colour filter.<br />
Measure absorbance against water for the reagent<br />
blanks, standards and test samples.<br />
Obtain collagen concentrations from the Standard<br />
Curve. Duplicates should be close to + 5% of their<br />
mean value.<br />
If sample absorbance values are at the top end of the standard curve the assay<br />
should be repeated and a set of higher concentration standards (15, 30 and 50µg)<br />
used, together with 1000µl of alkali reagent rather than 250µl. The assay is<br />
completed as above.<br />
READ SIRCOL MANUAL FOR SAMPLE PREPARATION DETAILS<br />
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