LDH Lab 5 & 6 Protocol

LDH Lab 5 & 6 Protocol - Section N TA: Josh LDH Lab 5...

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Section N TA: Josh LDH Lab 5 and 6 Protocol Materials LDH fractions, gel exclusion standards, pipettes, microcentrifuge tubes, syringe, gel exclusion apparatus, 1.5 mM pyruvate, 0.6 mM NADH, oxalate, oxamate, phosphate buffer Procedure 1) Obtain 100 ul of peak LDH column fraction from each group in a microcentrifuge tube and mix. 2) Obtain the gel exclusion standards from the TA. 3) Prepare sample by combining: -90 ul each of horse spleen ferritin, sweet potato b-amylase, human hemoglobin, bovine carbonic anyhydrase, horse heart cytochrome c -200 ul each of pooled LDH peak fractions and of gel exclusion column buffer (sodium azide is a poison) 4) Mix final sample by pipetting up and down. 5) Remove insoluble material by centrifugation (1 min) then filtering. Withdraw supernatant with a 1 ml syringe (no needle), then attach a syringe filter and slowly filter the contents into another microcentrifuge tube. 6) Make sure final volume is greater than 600 ul, return the standards and leftover LDH to TA, and discard the syringe and filter. 7) Start the BioLogic HR program, switch the “Inject Valve” to the “Purge” position. 8)
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This note was uploaded on 05/10/2008 for the course BIO CHEM 153L taught by Professor Kim during the Spring '08 term at UCLA.

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LDH Lab 5 & 6 Protocol - Section N TA: Josh LDH Lab 5...

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