Biochem Lab Final

SDS-PAGE purpose

Separates proteins by molecular weight to determine purity and size

How SDS works

Coats proteins with uniform negative charge

Purpose of heating in SDS-PAGE

Denatures protein structure

Reducing Agents (β-mercaptoethanol/DTT

Break disulfide bonds before electrophoresis

Migration in SDS-PAGE

Smaller proteins migrate faster toward the positive electrode

High % acrylamide gel

Small pores; used for small proteins

Low % acrylamide gel

Large pores; used for large proteins

Buffer components

Weak acid + conjugate base

Weak base + conjugate acid

Effect of salt on buffers

Affects protein stability/solubility

Protein pI (Isoelectric point)

pH where protein has no net charge

Protein charge when pH < pI

Protein is positively charged

Protein charge when pH > pI

Protein is negatively charged

Protein solubility at pI

Proteins may precipitate at their pI

Kinetic assays purpose

Measure rate of substrate

product conversion

Detectable assay signals

Absorbance or fluorescence change

Km definition

Substrate concentration at ½ Vmax

Km relationship to affinity

Low Km = high affinity

Specific activity definition

Enzyme units per mg protein

Fold purification equation

Specific activity after step / before step

Centrifugation purpose

Removes cell debris

Ammonium sulfate precipitation

Separates proteins by solubility

Dialysis purpose

Removes small molecules/salts

Chromatography types

Ion exchange, size exclusion, affinity

SDS-PAGE in purification

Checks protein purity at each step

Agarose gel electrophoresis purpose

Separates DNA fragments by size

DNA migration reason

DNA is negatively charged

Fragment size and migration

Small fragments migrate farther

DNA gel components

Agarose, buffer, loading dye, ladder, DNA stain

Ion exchange chromatography basis

Separates proteins by charge

Cation exchange resin charge

Negatively charged resin binds positive proteins

Anion exchange resin charge

Positively charged resin binds negative proteins

Protein elution method

Increase salt or change pH

Resin used for lysozyme

Sephadex CM (negatively charged)

Lysozyme charge at pH 8.2 (pI = 10.7)

Positively charged

Lysozyme binding/wash buffer

0.05 M Tris pH 8.2

Lysozyme elution buffer

0.2 M carbonate pH 10.5

Lysozyme extinction coefficient

ε = 37800

Lysozyme activity assay wavelength

450 nm

Lysozyme activity units

ΔAbs/min × 1000

Beer’s Law

A = ε × l × c

Dilution factor formula

DF = total volume / volume transferred

Actual concentration formula

Measured concentration * DF

Restriction enzyme function

Cut DNA at specific palindromic sequences

Sticky vs blunt ends

sticky ends have overhangs; blunt ends do not

Typical restriction enzyme temperature

37°C

Bradford assay

Turns blue when dye binds protein; measured at 595 nm

BCA/biuret assay

Copper-based assay; measured around 562 nm

UV 280 nm absorbance

Detects aromatic amino acids in proteins

Tyrosinase substrate

L-DOPA

Tyrosinase product

Dopachrome (measured at 475 nm)

Tyrosinase Part A variable

vary enzyme concentration

Michaelis-Menten equation

V = Vmax[S] / (Km + [S])

Lineweaver-Burk Equation

1/V = (Km/Vmax)(1/[S]) + 1/Vmax

Agarose needed for 40 mL gel

400 mg

Gel setting time

~30 minutes

Common units in biochemistry

mM, µM, nM, mg/mL, µg/mL, U/mL, OD units

Conversion between common units

1 mM = 1000 µM; 1 mg = 1000 µg; 1 mL = 1000 µL

Meaning of Ka

Acid dissociation constant; strength of an acid

Meaning of pKa

pKa = -log(Ka); lower pKa = stronger acid

Calculating final concentration after dilution

Final concentration = stock concentration/DF

Pipettor use

Pipettes dispense set volumes using disposable tips

Reading pipettor dial (P20)

Shows tens-ones-tenths of microliter (e.g., 052 = 5.2 microliter)

Reading pipettor dial (P200)

Shows hundreds-tens-ones of microliter (e.g., 125 = 125 microliter)

Reading pipettor dial (P1000)

Shows thousands-hundreds-tens of microliters (e.g., 100 = 1000 microliter)

pI definition

pH where a molecule has no net charge

Calculating pI for two pKa amino acids

pI = (pKa1 + pKa2) / 2

Henderson-hasselbalch equation

pH = pKa + log([A-] / [HA])

Acid:Base ratio from pH and pKa

[A-] / [HA] = 10^(pH-pKa)

Weak acid equation

pH = ½(pKa – log C)

Weak base equation

pOH = ½(pKb – log C)

% Saturation Equation (Ammonium Sulfate 1)

%S1 = ((S2 × V2) – (S1 × V1)) / (S100 – S1)

% Saturation Equation (Ammonium Sulfate 2)

Amount salt needed = (Sdesired – Scurrent) × V / constant

Reagents used in experiments

SDS, Tris buffer, ammonium sulfate, DTT, β-mercaptoethanol, carbonate buffer, SYBR Gold

Proteins purified in experiments

Lysozyme, tyrosinase (kinetics), albumin (standards)

Protein precipitation methods

Ammonium sulfate salting-out, pH precipitation at pI, organic solvent precipitation

Amino acid titration curve

Shows pH vs. added base; plateau at pKa values; midpoint = pKa; pI at average of relevant pKa’s