User:Z3284851

From CellBiology

--Z3284851 11:47, 1 August 2011 (EST)

ELISA Kits

(Not ordered - Human orosomucoid, ORM ELISA Kit CSB-E09393h AU$920.00+GST http://www.liferesearch.com/human-orosomucoid-orm-elisa-kit.html from Cusabio)


Life Research Pty Ltd ABN: 51 137 816 267 ADDRESS:Level 1, 5 Lakeside Drive Burwood East Victoria 3151 Tel: 1300 543 373 (+61 3 9803 0045) Fax: 03 8648 5841 Email: info@liferesearch.com

LDH ASSAY

REAGENT PREP 1. Assay Buffer- PBS Solution 2. LDH Diaphorase - Reconstitute 1ea vial LDH Diaphorase with 150μl of Assay buffer - Keep on ice during use, -20C when not in use 3.LDH Reaction Solution: TO make 10mL of reaction solution, add 100μL of each to 9.6ml Assay buffer -NAD+ (100x) - Lactic Acid (100x) - INT(100x) - Reconstituted diaphorase

ASSAY PROTOCOL

LDH STANDARD - Reconstitute contents of LDH standard in 1.8ml of PBS. Keep on ice during ise, standard unstabel- store at -80C - Obtain six clean test tubes, label #1-#6 - Aliquot 475μl of CSF (diluted) into #1 and 250μl into #2-#6 - Transfer 25μl of reconstituted LDH standard into tube #1, mix thoroughly. 1st point of standard curve- conc. 10mU/ml (10,000μU/ml) - Serially dilute standard, remove 250μl from #1 and place in #2, mix - Remove 250μl from #2 and place into #3, mix thoroughly - Repeat procedure for tubes #4-#5 - #6- Leave blank

PROCEDURE- PERFORMING THE ASSAY 1. Using a 96well plate, transfer 100μl of the standard prepared abobe into apropriate wells 2. Transfer 100μl of each CSF sample to the plate 3. Add 100μl of reaction solution to each well 4. Incubate the plate with gentle shaking on an orbital shaker for 30mins at room temp 5. Read absorbance at 490nm with a plate reader.

Experiment 1

CSF samples diluted 1600x with dilution buffer prior to assay A. 10μL CSF into 390μl of dilution buffer B. 10μl of A into 390μl of dilution buffer

Experiment 2

CSF samples diluted 10x with dilution buffer prior to assay - 30μl of CSF into 270μl of PBS