CLAB 485 and CLAB 486 Learning Goals Biochemistry Laboratory I ...

CLAB 485 and CLAB 486 Learning Goals
Biochemistry Laboratory I and II
November, 2009
Course Catalogue Descriptions:
485. Biochemistry Laboratory. Credit 1 hour. Prerequisite: Registration for or prior credit for
Chemistry 481. A laboratory to accompany Chemistry 481. Experiments are designed to
demonstrate the properties of amino acids, proteins, carbohydrates, lipids, and nucleic acids with
emphasis on enzyme kinetics and protein purification. Three hours of laboratory a week.
Laboratory fee: $30.00.
486. Biochemistry Laboratory. Credit 1 hour. Prerequisite: Registration for or prior credit for
Chemistry 482 and prior credit for Chemistry 485. A laboratory to accompany Chemistry 482.
Experiments are designed to demonstrate some of the major metabolic pathways with emphasis
on energy considerations and interrelationships of the pathways. It also emphasizes the flow of
genetic information through replication, transcription, and translation. Three hours of laboratory
a week. Laboratory fee: $30.00.
Course Delivery and Assessment:
Instructors are encouraged to present content in a manner that is consistent with the scope and
sequence in which it is presented in the required textbook. The following pages list the minimum
learning goals that have been recommended for CLAB 485 and CLAB 486. Instructors may add
topics but should not omit learning goals from this list. If the instructor chooses to add topics,
he/she is encouraged to provide students with adequate written resources. Course assessments
should reflect the learning goals listed, and should include laboratory reports (number
determined by individual faculty), one midterm exam and one final exam.

CLAB 485 Learning Goals
Biochemistry Laboratory I
Pipettors and standard curves
• To understand and use P-20, P-100 and P-1000 micropipettors by repetitive pipetting
• To make dilutions of p-nitrophenol and perform spectroscopic analysis to identify the concentration of
an unknown
• To gain experience with the UV/VIS microplate reader
Buffers and buffering capacity
• To make solutions containing multiple compounds
• To make a buffer solution at a specific pH using the Henderson-Hasselbalch equation
• To investigate the ability of a buffer solution to withstand the addition of acid and/or base
• To determine the theoretical change in pH of a buffer solution upon the addition of acid and/or base
using the Henderson-Hasselbalch equation
• To investigate the relationship of pK to buffering capacity of acetic acid
Amino acids: Titration, pK, ion exchange and pI
• To relate the concept of pK to biological molecules (amino acids and proteins)
• To determine the pK values of an amino acid by performing an acid/base titration
• To separate amino acids using the process of paper chromatography to identify a dipeptide
• To contrast the ninhydrin reaction for primary and secondary amines
• To compare the spectral scans of aromatic amino acids in the UV range
• To separate amino acids via ion exchange chromatography and predict the elution order with increasing
pH
• To determine the pI of an amino acid
Beer-Lambert Law and proteins assays
• To demonstrate the relationship between absorbance and concentration using extinction coefficients
• To compare and contrast the Bradford and BCA methods of protein quantification
• To generate standard curves using bovine serum albumin as a protein
• To determine the concentration of a protein using the Bradford and BCA methods for protein assays
Horseradish peroxidase: Affinity chromatography, gel filtration, and polyacrylamide electrophoresis
• To understand the concept of lectins as they relate to horseradish peroxidase
• To purify horseradish peroxidase using a Concanavalin A affinity column
• To demonstrate the concept of competitive elution of a protein from an affinity column
• To collect fractions and determine absorbance at various wavelengths
• To perform a enzyme-coupled assay using ABTS as an electron donor
• To use extinction coefficients in the conversion of Δabs/min to μmol/min (rate)
• To perform the calculations necessary (activity, specific activity, total activity, yield, and fold
purification) for the creation of a protein purification table
• To create column profiles consisting of double y-axis graphs
• To determine the molecular weight of horseradish peroxidase using gel filtration
• To understand the concept of SDS-Polyacrylamide Gel Electrophoresis and determine an accurate
molecular weight for horseradish peroxidase
Enzyme kinetics: K m , V max , inhibition, K i , and substrate specificity
• To experimentally relate Michaelis-Menten graphs to Lineweaver-Burk graphs
• To understand the concept of reversible enzyme inhibition

• To develop an enzyme assay to determine the concentration of enzyme appropriate for a specified
Δabs/minute
• To develop an enzyme assay to determine the concentration of inhibitor appropriate for a specified
reduction in the rate
• To create an assay sufficient to provide both Michaelis-Menten and Lineweaver-Burk plots
• To experimentally determine the values of K m and V max using the generated data
• To experimentally show substrate specificity given both D- and L-isomers of a substrate
• To experimentally determine the type of irreversible inhibition and the value of K i

CLAB 486 Learning Goals
Biochemistry Laboratory II
Nucleotides, hyperchromic effect, and DNA melting
• To investigate the UV spectroscopy of nucleotide bases in RNA and DNA
• To investigate the hyperchromic effect of dsDNA
• To investigate the effect of heat on dsDNA
• To investigate the effect of DNase on DNA structure
Plasmid DNA Isolation
• To investigate the effects of SDS on cell membrane rupture and cell lysis
• To purify plasmid DNA from lysed cells using silica column
• To determine the concentration of plasmid DNA using UV spectroscopy
Restriction Analysis and Gel Electrophoresis of Plasmid DNA
• To use three endonucleases to cleave plasmid DNA and control DNA
• To separate digests relative to native plasmid DNA by gel electrophoresis, including pouring agarose
gels.
• To analyze the size of DNA and DNA fragments
• To predict the size of DNA fragments and the agreement between theory and practice.
Polymerase Chain Reaction (PCR) and Amplification of p-Glo Gene
• To use Thermus acquatitus DNA polymerase to amplify the pGlo gene.
• To purify amplified DNA
• To analyze amplified DNA by agarose gel electrophoresis and endonuclease mapping.
pGlo Expression
• To transform a pGlo plasmid into E. coli
• To investigate the effects of arabinose and ampicilin on growth of pGlo-transformed and control E. coli
• To investigate the effects of arabinose and ampicilin on and expression of GFP in transformed cells.
Green Fluorescent Protein (GFP) purification and polyacrylamide gel electrophoresis
• To prepare overnight culture of E. coli transformed with pGlo
• To lyse E. coli enriched in GFP-enriched cells.
• To purify GFP by hydrophobic interaction chromatography
Polyacrylamide Gel Electrophoresis and Western Blot Analysis of Green Fluorescent Protein
• To determine the relative molecular mass of GRP using SDS PAGE
• To identify the presence of GFP using western blot analysis of SDS PAGE gels
Glycolysis –Design, Preparation, and Analysis (3 labs)
• To design experiments to prove the final three steps of the glycolytic pathway.
• To prepared reagents for these designed experiments
• To prove experimentally the last three steps of glycolysis
Allosteric control of isocitrate dehydrogenase I
• To design an assay for isocitrate dehydrgenase from yeast.
• To generate dose-response curves for IDH in the absence and presence of allosteric activators and
inhibitors
• To analyze the effects of allosteric activators and inhibitors on IDH Km and Vmax kinetic constants.