(n)
0.1MSodiumPhosphateBuffer:Weigh 6.10g of
sodiumdihydrogenphosphate
dihydrate and 10.87g of disodium hydrogenphosphate dihydrate into a1L volumetric flask.
Dissolve inwater andmakeup tomark. Adjust pH to 7with 2M sodium hydroxide.
(o)
0.1%Phosphoric acid:
In a1L volumetric flask, add500mLwater. Add 1.2mLof ortho-
phosphoric acid.Mix andmakeup tomarkwithwater.
StandardSolutions
(a)
Stock standard (100µg/mL):Weigh 25mgof biotin referencematerial into a250mL
amber volumetric flask. Add 150mLof water and shake onan orbital shaker for 24 hours.
(b)
Intermediate standard (100µg/100mL): Dilute1mL of the stock standard to100mL
withwater.
(c)
Calibration standards (CS) 1-10µg/100mL: Prepared as follows
CS 1 (1.0µg/100mL): Dilute 100µLof intermediate standard to 10mLwithwater.
CS 2 (2.5µg/100mL): Dilute 250µLof intermediate standard to 10mLwithwater.
CS 3 (5.0µg/100mL): Dilute 500µLof intermediate standard to 10mLwithwater.
CS 4 (7.5µg/100mL): Dilute 750µLof intermediate standard to 10mLwithwater.
CS 5 (10µg/100mL): Dilute 1mL of intermediate standard to 10mLwithwater.
SamplePreparation
The samplewasweighed into a100mL amber glass screw cap bottle; 50mL of 0.1M sodium
phosphate buffer was added andmixed on a vortexmixer for 30 seconds. Autoclaved the
sample preparation at 121
0
C for 25min and cooled to room temperature. Quantitatively
transferred the extracts into a100mL volumetric flask andmade up to volumewith 0.1M
sodium phosphate buffer. Theextract was transferred into a centrifuge tube and centrifuged
the samples at 4000rpm for 15min. The supernatant was filteredusing theWhatmanglass
microfiber filter paper (re-filtered the samples if the filtratewas cloudy). The immunoaffinity
BIO-02
FORWORKINGGROUP/ERPUSEONLY
DONOTDISTRIBUTE
