Biochemical Techniques and Methods Flashcards

In enzyme kinetics, the Michaelis constant (Km) is defined as:

Answer: The substrate concentration at half-maximal velocity

Km is the substrate concentration at which reaction velocity equals Vmax/2; it is a measure of substrate affinity (lower Km = higher affinity) but is not a true equilibrium dissociation constant.

Competitive inhibition of an enzyme:

Answer: Increases apparent Km without changing Vmax

A competitive inhibitor competes with substrate for the active site, increasing apparent Km (reduced affinity) but leaving Vmax unchanged because excess substrate can overcome inhibition.

Gel filtration (size-exclusion) chromatography separates proteins such that:

Answer: Larger proteins elute first

Larger proteins cannot enter the pores of the gel matrix and pass through the column faster (elute first), while smaller proteins enter the pores and take longer to elute.

Reverse transcriptase PCR (RT-PCR) begins with which step?

Answer: Conversion of mRNA to complementary DNA (cDNA)

RT-PCR starts by using reverse transcriptase to synthesize a complementary DNA (cDNA) copy of the mRNA template, which can then be amplified by conventional PCR.

The Bradford assay measures protein concentration using which dye?

Answer: Coomassie Brilliant Blue G-250

The Bradford assay uses Coomassie Brilliant Blue G-250, which shifts its absorbance maximum from 465 nm to 595 nm upon binding to proteins; the absorbance at 595 nm is proportional to protein concentration.

Southern blotting detects:

Answer: Specific DNA sequences

Southern blotting (named after Edward Southern) involves electrophoresis of restriction-digested DNA, transfer to a membrane, and hybridization with a labeled DNA probe to detect specific sequences.