LAB 10 Proteolysis SDS Page and RP HPLC

proteolysis

• process breaks down proteins or peptides into amino acids by action of enzymes

• protease enzyme hydrolyze peptide bond to break it into smaller fragments

• Non-enzymatic proteolysis occurs with use of mineral acids & heat

proteolysis

schematic representation

figure shows basic chemical structure of protein’s 2 amino acids, connected by peptide bond (represented by dotted line in figure). It’s at peptide bond that proteinase, with addition of water (H₂O), cuts protein into 2 fragments.

trypsin

• serine protease produced in pancreas, where it hydrolyses proteins

• excreted as inactive protease trypsinogen

• cleaves peptide chains at carboxyl side of amino acids lysine or arginine, except when followed by proline

trypsin 2

• uses special serine amino acid in protein-cutting rxn, serine protease

• serine proteases are diverse family of enzymes, all use similar enzymatic machinery

• Trypsin & chymotrypsin that proteolyze proteins during digestion

• Each has particular taste for protein chains: trypsin cuts next to lysine & arginine, chymotrypsin cuts next to phenylalanine & other large amino acids

protein separation

• SDS PAGE Electrophoresis is 1 of 3 major techniques used to analyze & purify proteins (other 2 are chromatography & centrifugation)

• matrix (stationary phase) is made of acrylamide polymer

• By controlling % (from 3% - 30%) of polyacrylamide, precise pore sizes can be obtained for separating protein from 5 - 2,000 kDa

protein separation

details

• Electric current provides force for migration

• gel is run in running buffer containing electrolytes

• Sodium dodecyl sulphate (SDS) is added to running buffer to mask charges

• SDS-polypeptide complexes have same neg charge & shape & migrate through gel according to polypeptide size

• Once individual proteins are separated, gel is stained with Coomassie blue (GelCode Blue) to visualize protein bands

protein separation

laemmli buffer

• designed to dissociate proteins into individual polypeptide subunits

• Sodium dodecyl sulfate (SDS) is ionic detergent which denatures protein complexes by wrapping around polypeptide backbone

• Dithiothreitol (DTT) is thiol reagent that cleaves disulfide bonds

• When protein sample is heated in presence of DTT & excess of SDS, proteins are fully dissociated into their subunits

protein separation

laemmli buffer

5X Lammeli sample buffer with DTT  (200 mM)

• Contents: 20% SDS 6 ml

• Bromophenol blue 6 mg

• Glycerol 4.7 ml

• 1M Tris pH 8 0.6 ml

• DTT (per 5 ml of Laemmli buffer) 0.465 g

visualization of proteins

• Protein bands are stained with dye to observe

• Proteins are stained using a Gel Code Blue

• GelCode Blue Safe Protein Stain is proprietary Coomassie blue based stain

• Gel Code Blue stains gel which can be easily washed off by rinsing repeatedly with water

Note: coommassie blue is also used in coomassie blue protein assay (bradford assay)

videos

protein molecular marker

• contains mixture of proteins of known molecular weight & loaded on gel to estimate size of proteins in sample

• In today’s lab, “Precision Plus Protein Standard” containing range of protein sizes is used to size GFP

stationary phase & mobile phase

stationary phase

• aka ‘matrix’

• thin layer on solid support (glass/plastic) or packed in column

• compounds are separated based on relative affinity to stationary & mobile phase

• If compound has greater affinity for mobile phase as compared to stationary phase, compound will move with mobile phase

• As a result, each compound in sample mixture moves at diff speeds through stationary phase

stationary phase & mobile phase

stationary phase

normal phase chromatography

• has more polar stationary phase & made of silica gel

• Polar compounds bind strongly to matrix while hydrophobic compounds move quickly

• Silica gel is acidic & offers poor separation of basic samples & causes deterioration of acid-labile molecules 

stationary phase & mobile phase

stationary phase

reversed phase chromatography

• matrix particles (silica gel) coated with compounds with hydrophobic side chains (C18 chains)

• matrix has higher affinity for hydrophobic compared to polar compounds

• highly polar compounds from sample easily eluted using polar mobile phase (eluting solvent)

• For separation of charged proteins & compounds, silica molecules are attached with charged functional groups

stationary phase & mobile phase

stationary phase

ion-exchange chromatography

• has charged matrix, which binds to molecules with opposite charge

• Negatively charged proteins will bind to positively charged columns and vice versa

mobile phase

• liquid or gas & moves through stationary phase via capillary action or pressure

• 2 or more solvents (polar/nonpolar) can be mixed together in varying proportions to obtain best polarity (or pH) for separation

• Common mobile phases used in liquid chromatography are water (polar solvent), ethanol & methanol (less polar solvents)

• For more sophisticated chromatographic separations, other organic solvents (aniline, octane) are used

how to read a chromatogram

word "chromatogram" means a plot obtained via chromatography. See figure below

video

how to read a chromatogram

• chromatogram is 2d plot with vertical axis showing conc in terms of detector signal intensity & horizontal axis representing analysis time

• When no compounds are eluted from column, a line parallel to horizontal axis is plotted → called baseline

• detector responds based on conc of target compound in elution band

• obtained plot is more like shape of bell rather than a triangle = shape is “peak”. 

how to read a chromatogram

retention (tR)

• time interval between sample injection point & apex of peak

• required time for non-retained compounds (compounds with no interaction for stationary phase) to go from injector to detector is dead time (t0).

how to read a chromatogram

peak height h

• vertical distance between peak's apex & baseline, & peak area (A) colored in light blue is area enclosed by peak & baseline

• results will be used for qualitative & quantitative analysis of sample's components

high performance (or pressure) liquid chromatography (HPLC)

• Reverse phase HPLC with a C18 column used to separate samples using methanol

• mobile phase is water: methanol mixture. This equipment will be demonstrated to you by your instructor. 

• Dual pump system & computer software (method) is used to provide varying concs (gradients) of water & methanol during each run (see below)

• spectrophotometer is set to read at 254 nm wavelength. 

high performance (or pressure) liquid chromatography (HPLC)

instrumentation

solvent reservoir

Stores the solvent (mobile phase). 

high performance (or pressure) liquid chromatography (HPLC)

instrumentation

pump

High-pressure pump that generates a specific flow rate of mobile phase. 

high performance (or pressure) liquid chromatography (HPLC)

instrumentation

sample injector

valve in which you inject your sample

• Introduces sample into stream of mobile phase that will carry sample to HPLC column. 

high performance (or pressure) liquid chromatography (HPLC)

instrumentation

HPLC column

• Stationary phase of HPLC

• Contains packing material needed to separate injected sample

• For today’s lab, column consists of silica coated in C18 chains. 

high performance (or pressure) liquid chromatography (HPLC)

instrumentation

detector

• Internal UV-Vis spectrophotometer used to visualize separated compounds as eluted from HPLC column

• Sends info to computer data station. 

high performance (or pressure) liquid chromatography (HPLC)

instrumentation

computer data station

• Records electrical signal from detector & generates chromatogram

• computer data station is also used to identify & quantify conc of sample

high performance (or pressure) liquid chromatography (HPLC)

instrumentation

waste

Collects mobile phase that’s eluted from column. 

TF HPLC PROTOCOL

GFP CHROMATOGRAPHS

CHROMATOGRAMS

SDS PAGE GELS