electrophoresis for pgs by dr siva kumar reddy
TRANSCRIPT
Dr. M. Siva Kumar Reddy MD
ELECTROPHORESIS
common Indications for electrophoresis
Multiple myeloma *a sharp distinct M band appears in -globulin fraction* Acute infections *α1 and α2 globulins are increased* Nephrotic syndrome *decreased albumin with sharp and prominent α2
globulin* primary immune deficiency *Diminished -globulin band* α1-Anti trypsin deficiency *diminished α1-globulin band*
Other indications in clinical chemistry
Waldenstrom’s macroglobulinemia Primary amyloidosis Unexplained peripheral neuropathy New onset anemia with renal failureHypercalcemia related to malignancyRouleaux formation on peripheral smear renal insufficiency with associated
serum protein elevation.
Unexplained pathologic fracture or lytic lesion on radiograph...
CNS infections and inflammationsDyslipidemiasEpidemics (micro organism causing
epidemic can be detected using PFGE)Drug monitoring using capillary
electrophoresis
Other indications in clinical chemistry
INTRODUCTION
Electrophoresis is a physical method analysis which involves separation of the compounds that are capable of acquiring electric charge in conducting electrodes.
DEFINITION
Electrophoresis may be defined as the migration of the charged particle through a solution under the influence of an external electric field.
TYPES OF ELECTROPHORESIS
ELECTROPHORESIS
ZONE ELECTROPHORESIS
MOVING BOUNDARY ELECTROPHORESIS
ZONE ELECTROPHORESIS
PAPER ELECTROPH
ORESIS
WHATMANN FILTER PAPER ELECTROPHO
RESIS
GEL ELECTROPH
ORESIS
CAPILLARY ELECTROPH
ORESIS
GEL ELECTROPHORESIS
AGAROSE GEL ELECTROPHORESIS
POLYACRYLAMIDE GEL ELECTROPHORESIS
SDS-PAGE
DISC ELECTROPHORESIS
ISOELECTRIC FOCUSSING
IMMUNOELECTROPHORESIS
TWO DIMENSIONAL ELECTROPHORESIS
MOVING BOUNDARY ELECTROPHORESIS
NO SUPPORTING MEDIUM USED FOR MOVING BOUNDARY ELECTROPHORESIS
Arne Tiselius (10 August 1902 – 29 October1971) Swedish biochemist
ZONE ELECTROPHORESIS
Basic components of zone electrophoresis…..
supporting medium. Buffer chamber. Power pack and electrode. Sample dissolved in buffer tracking dye. Perspex cover to encase the entire
chamber.
Buffers
Most commonly used buffers in zone electrophoresis.
*ACETATE
*PYRIDINE
*BARBITONE
*TRIS(2-Amino,2-hydroxy methyl,1-3diol) most commonly used
*CITRATE
Buffers at a glance……..
Barbitone buffer(pH-8)• s.Protein
separation• Poor
resolution• Week buffer
Phosphate buffer(pH7)• Enzymes
separation• Low buffering
capacity• High
conductivity
TRIS- BORATE –EDTA(TBE)(pH-8)• Nucleic acid
separation• Good resolution
&high buffering capacity
• Low conductivity
Buffers at a glance………
TRIS-acetate-EDTA(pH-8) (TAE)• Nucleic acid
separation• High resolution &
high buffering capacity
• Low conductivity
TRIS- glycine buffer(pH >8)• Protein separation• High buffering capacity• Low conductivity
Preparation of Buffer
TRIS- GLYCINE buffer
In 600 ml
distilled water
3.6grams TRIS
4.6 grams
GLYCINE
PROCEDURE1 • Sample is spotted on supporting
medium
2 • Medium is placed between electrodes
3 • Power pack is switched on
4• Voltage applied till dye has
travelled approx 2cm from other end
5• Medium removed and dried• stained with coomassie brilliant
blue
6 • Destained with 5% acetic acid
7 • Bands are visualized and analyzed using densitometer
Factors influencing electrophoresis
Supporting
medium
Buffer
Electric
current
Size,shape &net charge of molecule
Heat and electroendosmosis
Paper electrophoresis
Paper electrophoresis is mainly used for separation of protein(at low voltage), amino acids and nucleotides(at high voltage).
Greatest disadvantage of paper electrophoresis is diffusion, adsorption and electroendosmosis.
To over come these issues high voltage is applied 10,000 volts that results in good resolution and rapid separation.
Small molecules like amino acids and nucleotides are separated by high voltage electrophoresis
Paper electrophoresis
Cellulose acetate Electrophoresis
General procedure is similar to that of paper electrophoresis
Cellulose acetate electrophoresis has replaced paper
electrophoresis
Better resolution in short time.
Negligible adsorption and EEO
Suitable for fractionation of polysaccharides and lipoproteins
Bands are easily detected due to transparency of medium.
Cellulose acetate Electrophoresis
Pulsed Field Gel electrophoresis
Separation of DNA using constant electric field is called constant field gel electrophoresis(CFGE).
Separation of DNA altering the strength and direction of electric field between the electrodes is called as pulse field gel electrophoresis(PGFE)
PGFE used to separate high molecular weight DNA.
CFGE is normal agarose gel electrophoresis for molecular weight up to 500kb.
PGFE used to separate human chromosome and yeast chromosome with sharper bands and high resolution.
PFGE
SDS PAGE….
Polyacrylamide gels are used for protein fractionation in the presence of SDS(sodium dodecyl sulphate)
Polyacrylamide could be used in the form of tube gel(for small volume of samples)
Vertical slab gel (for large volume of samples) Denaturing of protein sample is needed because protein structure is stable
with hydrophobic interaction, hydrogen bond, and disulphide bond. Stable structure does not favour electrophoresis and need to disturb its
structure Denaturing agents SDS, urea, and beta mercaptoethanol
polyacrylamide gel electrophoresis protein electrophoresis/ SDS PAGE
PROCESS OF SDS PAGE
Denatured
sample loaded
Running buffer(TRIS-
GLYCINE) ph8.8
80volts current
applied& allow it till
it reach 5cm above lower end
Staining by
coomassie or silver staining
Destaining using
methanol, acetic acid, water
mixture
SDS PAGE Applications
Determine molecular weight of proteinsTo fractionate protein subunitsTo assess the purity of proteins samples
Native PAGE with out SDS To separate enzymes and
isoenzymes
Disc electrophoresis (tube electrophoresis)
Disc electrophoresis(tube electrophoresis)
Tank of 100 ml buffer forms anode above and another tank of 100 ml forms cathode below.
Six holes of 2mm diameter are drilled1.5 cm apart and 1mm above the buffer.
Any protein specimen can be fractioned using this apparatus.
The order of resolution is prealbumin, albumin , postalbumins , ceruloplasmin ,transferrin, post transferrin fractions and gamma- globulins.
Lipoproteins separation and studies on Isoenzymes were also carried out using this apparatus.
Disc electrophoresis (tube electrophoresis)
Iso electric focusing
IEF is a type of gel electrophoresis in which compounds are separated in a gel with ph gradient , based on differences in iso electric point.
IEF gel has pre selected pH range.Compounds with pH less than iso electric pH
acquire negative charge and move towards anode and vice versa move towards cathode.
Migration continues till the molecules reach a pH region in the gel, which is equal to their iso electric point.
At that point molecules become stationary and exist as zwitter ions.
Isoelectric focusing
IEF applications
To determine iso electric point of protein.
To separate iso enzymes.
To fractionate proteins with higher resolution.
To separate all amphoteric substances.
To study mono,di,tri substituted derivatives of
proteins.
2D PAGE
In 2D PAGE, the molecules are first separated by IEF in a gradient gel, and then by SDS PAGE in a second gel.
2D PAGE IS USED: * To resolve proteins and enzymes with high resolution *To assess purity of proteins.
2D PAGE
Molecules separated by…• pH gradient
gel cast on narrow cylindrical tube
Gel extruded from tube…• Incubated in
SDS for 30 min to denature the protein
Placed on second gel and stacking
gel, voltage applied and
protein sub units are separated
Immunoelecrophoresis
Rocket electrophoresis
IEF combines sensitivity of gel electrophoresis and specificity of immune reaction.
This technique is used to quantify antigen.
Capillary electrophoresis In capillary electrophoresis, separation
is done with in capillary tube under high pressure and high voltage to give better resolution in short time
Capillary electrophoresis applications
To separate amino acids.
To separate oligonucleotides and nucleic acids.
To separate drugs.
To separate metal ions.
To separate enantiomers.
Agarose Gel Electrophoresis
Is used for fractionation of both DNA and protein
But with different buffers and at different current and voltages.
Agarose gel is used as supporting medium which is a polysaccharide derivative of agar.
The pores of agarose gel act as molecular sieve.
Pore size of gel is inversely proportional to the initial concentration of agarose
0.8% of agarose used for DNA of 0.5-10kb.
Process of gel electrophoresis
1% AGAROSE 100MG WEIGHED AND TAKEN IN TO TEST TUBE
To the agar add 10 ml of TRISS-GLYCINE ……..
Place the test tube in boiling water bath for about 10minutes
After taking out from water bath evenly mixed agarose gel taken in to pipette…….
Pour evenly on to the slide using pipette
Serum+ bromophenol blue
Place the sample on the slide
POUR THE BUFFER IN TO THE TANK and connect wicks
Run power pack for 1.30 hours at 150volts for better results
Fixative (methanol or acetic acid)
For a period of 30 minutes leave the slide in the fixative
Drying of slide
Can use drier or hot air ovenDry the slide till the agarose fixes to slide
Preparation of COOMASSIE BLUE dye
Made to 100ml (CBB dye)
Glacial acetic
acid 7ml
Dye 300mg
Ethanol 50ml
Staining with coomassie blue
Leave the slide in the CBB stain for 30 minutes
Distaining with 5% acetic acid
Examine the slide for the bands
Normal serum pattern
Nephrotic syndrome
Analyze the slides using densitometer
Normal eletrophoretic pattern…
Inflammation vs normal
Normal vs multiple myeloma
Lipoprotein electrophoretic pattern…..
Hemoglobin electrophoretic abnormal patterns….
CSF Electrophoresis bands
Serum protein electrophoresis vs urine protein electrophoresis
Analysis of patterns of albumin………
Increased albumin Decreased albumin
• dehydration • Chronic cachectic or wasting diseases
• Chronic infections
• hemorrhage
• burns• Protein loosing
enteropathies• Impaired liver function
• malnutrition
• Nephrotic syndrome
Analysis of patterns of α1 globulins..
Increased α1 globulins Decreased α1 globulins
pregnancy α 1 antitrypsin deficiency
Analysis of patterns of α2 globulins ……… Increased α2 globulins Decreased α2 globulins
Adrenal insufficiency malnutritionAdrenocorticosteroid therapy
Megaloblastic anaemia
Diabetes mellitus Protein losing enteropathy
Nephrotic syndrome Severe liver diseaseWilson’s disease
Abnormal patterns of β1 or β2 globulins..
increasedβ1 or β2 globulins
decreasedβ1 or β2 globulins
Biliary cirrhosis Protein malnutritioncarcinomasCushings diseaseDiabetes mellitushypothyroidismIron deficiency anemiaMalignant hypertensionnephrosisPoly arteritisObstructive jaundice
Abnormal pattern of -globulins…… Increased -globulins
Decreased -globulins
amloidosis Agamma globulinemia Chronic infections hypogammaglobulinemia Chronic lymphocytic luekemia cirrhosis Hodgkin’s diseases Malignant lymphoma Multiple myeloma Connective tissue disorders Waldenstrom’s macroglobulinemia