Sircol Assay Manual - Biocolor Assays

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