ASSIGNMENT
ON
2- DIMENSIONAL
ELECTROPHORESIS
Compiled by: APARNA G SINGH
MSC PART (1) NEURO
DEPARTMENT OF LIFE
SCIENCES
INDEX
Introduction:
WHAT IS ELECTROPHORESIS?
TYPES OF ELECTROPHORESIS?
GEL ELECTROPHORESIS?
2-D ELECTROPHORESIS
MATERIALS AND METHODS
PROTOCOL
APPLICATIONS
Conclusion
INTRODUCTION
What is electrophoresis?
Electrophoresis is defined as a technique wherein molecules get separated based on their charge, size, gradient or binding affinity
It was first observed by Ferdinand Fredric Reuss in the year 1807 where he noticed that on the application of constant electric charge made clay particles dispersed in a fluid medium (in this case water) migrate. Its mainly caused by the presence of a charge gradient between the surface of particle and the surrounding fluid medium. it forms the basic strata for many bio analytical techniques used in biochemical reactions.
Electrophoresis of positively charged cations is known as cataphoresis wherein that of the negatively charged ones in known as anaphoresis.
The technique applies a negative or positive charge to the respective molecule so that it travels towards the opposite pole an that's how the separation is seen.
PAGE (Polyacrylamide gel electrophoresis) has a more clear and better resolution as compared to agarose and is more suitable for quantitative analysis. This technique also has its applications in plasmid analysis which helps us to understand how bacteria becomes resistant to a specific antibiotic.
TYPES OF ELECTROPHORESIS:
1.Affinity electrophoresis
2.capillary electrophoresis
3.Immunoelectrophoreisis
4.Istachoelectrophoresis
5.Gel electrophoresis
6. Dielectrophoresis
WHAT IS GEL ELECTROPHORESIS:
Gel electrophoresis is a type of electrophoresis wherein macromolecules such as (DNA,RNA, Proteins) get separated on the basis of their size and charge which can be further analysed. It has great applications in clinical chemistry wherein its used to separate proteins by charge/size and in molecular biology to separate DNA /RNA fragments.
Nucleic acid molecules are separated from eachother by applying an electric field around them that enables the negatively charged molecules move through a matrix made up of agarose /other materials. It is size dependent hence smaller the molecule ,faster it moves and longer and heftier the molecule ,slower it migrates.theis is because of the fact that smaller molecules are able to migrate through the pores of the gel .this phenomenon possessed by the molecules in their migration is know as 'Sieving'
Protein molecules are separated on the basis of charge in an agarose matrix because the pore size the the gel are too huge to sieve proteins through them.Gel electrophoresis can also be used to separate nanoparticles.
Gel electrophoresis uses gel as an anticonvective/sieving medium for the movement of charged particles in an electric fiels .Gels suppress the thermal convection currents caused due to the application of electric field during electrophoresis and acts as an sieving medium.
DNA Gel electrophoresis is usually done for analytical purpose ofthen after DNA amplification is done by PCR technique but it may also be used as an preparative method before the use of other methods such as mass spectroscopy, RFLP(Restriction Fragment Length Polymorphism),PCR (Polymerase Chain Reaction), Cloning, DNA sequencing, Southern blotting.
2- DIMENSIONAL GEL ELECTROPHORESIS
2-d electrophoresis is a powerful and widely used technique for protein complex analysis from given biological samples .
This technique separates proteins according to two independent properties in 2 important steps:
The 1st dimensional step wherein isoelectric focusing (IEF) separates proteins according to their isolelectric points and t
2-dimensional step wherein sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) separates protein molecules based on their molecular masses.each spot formed on the resultant 2-D get corresponds to a single protein in the given sample .hence, thousands of different proteins can be separated from eachother and their molecular weight and amount can be estimated .
2- D electrophoresis was first introduces by O'Farell in the year 1975 which as updated version of the technique ,the original technique made use of an carrier ampholyte containing polyacrylamide gels cast in narrow tubes
REQUIREMENTS :
Buffer
7M Urea
2M Thiourea
4% CHAPS
Add fresh: 20mM Spermine 20mM DTT 1mM PMSF
Rehydration Buffer
8M Urea
2% CHAPS
Add fresh: 20mM DTT 0.5% IPG Buffer
trace amount of bromophenol blue
Equilibration Buffer
6M Urea
30% Glycerol
2% SDS
50mM Tris-HCl pH 8.8
EQ Buffer 1 ' add fresh 1% DTT
EQ Buffer 2 ' add fresh 2.5% Iodoacetamide
Preparation
Remove media from flasks.
Add 4mL of trypsin to each flask and place back in incubator for 2-3 minutes.
Remove flasks from incubator and add 4mL of media to each flask to inactivate trypsin.
Collect cells into 15mL conical tubes and spin @ 3K RPM for 5-10 minutes at RT.
Remove supernatant from conical tubes and add 5mL of 1X PBS to each tube.
Resuspend cells in 1X PBS in order to remove excess trypsin and media.
Spin @ 3K RPM for 5-10 minutes at RT.
Remove supernatant from conical tubes and add 1mL of fresh lysis buffer to each tube.
Resuspend cells in fresh lysis buffer then place tubes in refrigerator at 4''C for 2-4 hours.
Transfer cell lysates into 1.5mL microcentrifuge tubes (Beckman tubes) and spin at 40K RPM for 1 hour at 4''C.
Carefully remove supernatant and transfer to clean microcentrifuge tube.
Determine protein concentration and store remaining lysate in -80''C freezer.
Isoelectric Focusing/The First Dimension
Prepare 2.5mL of fresh rehydration buffer for each type of DryStrip.
Place samples on ice to thaw and collect 1.5mL microcentrifuge tubes for sample dilution.
Remove the necessary number of DryStrips from -20''C freezer.
Determine the amount of rehydration buffer necessary for strip length.
Add 200-300ug of cell lysate to clean microcentrifuge tubes and add the difference between the total volume and the volume of sample using fresh rehydration buffer.
Remove plastic cover from strip holder and set aside.
7) Remove protective cover from Dry Strip before placing gel side down into strip holder starting at the anode (pointed end) and laying strip down to the cathode (blunt end).
Move strip back and forth in order to spread out rehydration buffer.
Make sure that all bubbles are removed from underneath the DryStrip before adding DryStrip Cover Fluid (Mineral Oil).
Add cover fluid from one end until it reaches the middle of the strip holder then add from the opposite side so that fluid meets in the middle.
Complete the same process for all strips then place the strip holders on IPGphor system.
Set up protocol for the length strip that is being used.
For 13cm strip:
Rehydration at 20''C for 12 hours 50uA per strip
500V for 1 hour
1000V for 1 hour
8000V for 3-5 hours
15)Move onto equilibration step or rinse strip with MilliQ water and place into screw cap tubes for storing in -70''C freezer.
Equilibration
Prepare 15 mL of fresh equilibration buffers 1 & 2 for each strip.
Wash strips with MilliQ water before placing into equilibration buffer 1. If you are removing strip from -70''C, let tube sit on lab bench to thaw strip. When strip is thawed (strip will be clear) place into equilibration buffer 1.
Incubate in equilibration buffer 1 for 15 minutes.
Remove strip and rinse with MilliQ water before placing into equilibration buffer 2.
Incubate in equilibration buffer 2 for 15 minutes.
Remove strip and rinse with MilliQ water and place strip on its side on filter paper to allow excess water to drain from strip.
Running the Second Dimension
Prepare gel by removing water saturated butanol from top of gel and washing with 1X running buffer.
Cover top of gel with 1X running buffer and lay strip across the top of the gel making sure that the gel is lying flush with the gel and remove any bubbles between the strip and the top of the gel.
Remove the running buffer and add warm 1% agarose made with 1X running buffer.
Allow the agarose to cool and solidify, which should only take a few minutes, before moving to the electrophoresis apparatus.
Add 1X running buffer to the upper and lower buffer chambers and place gel inside apparatus.
Run gel for 15 minutes at 10mA per gel then run gel at 25mA per gel for 4-6 hours (until BPB band reaches bottom of gel
Polyrylamide Gel Electrophoresis
Gel type
Sample volume(''l/well)
Total well volume(''l/well)
Mini Gels 0.75 mm, 10 well
2 to 5
25
Large Gels 0.75 mm, 10 wells
10-140
150
Sample buffer
0.062 M tris-HCl, pH6.8
2% SDS
0.01% Bromophenol Blue
10% Glycerol
5% 2-mercaptoethanol
Upper buffer
10% SDS
10 mL
250mM EDTA
4 mL
Reservoir buffer
100 mL
Nanopure water
add enough to 1 L mark
Lower buffer
Reservoir buffer
100 ml
Nanopure water
add enough to 1 L mark
Store buffers in refrigerator.
Sample Preparation
Prepare the sample with the sample buffer to a concentration of 1 ''g/''l.
Heat at 100 ''C for 2-3 min in a boiling water bath.
Sample loading
Remove the comb from the cassette by sliding it slowly with a steady motion straight up. Do not distort or tear any wells.
Flush the wells with water to remove residual acrylamide
For glass cassettes leave the tape on the side of the gels.
Use permanent marker to draw the bottoms of the wells. That will make it easier to load.
Insert these plates on the electrophoresis unit.
Pour the upper and the lower buffers
Load the appropriate amounts of samples. Load sample buffer in any wells that do not contain samples.
Gel Visualization
Copper Staining
Prepare the Copper stain by dissolving 4 g of CuCl2 in 100 mL of water.
Wash the gels with nanopure water.
Pour the Cu stain on the gel. Make sure that the gel is completely submerged.
Rock for 5 minutes.
Pour the stain into the waste and wash the gel with water to remove excess copper.
Store the gel in water in the refrigerator.
Zn Staining
Dilute Imidazole (Solution A) 1:10 with water, mix thoroughly. Dilute Zinc Sulfate (Solution B) 1:10 with deionized water, mix thoroughly. Gels will require 100-150 mL so that they are compeletely immersed.
Place the gel in a staining container and add Imidazole, solution A. Rock at room temperature for 10 min.
Decant Solution A, and add the dilute Zinc sulfate, solution B. Make sure the gel is completely covered to ensure even staining. Rock at room temperature for 30 sec approx while the gel develops.
Decant solution B, and add 100 mL of water. Rinse for 3-5 min rocking at room temperature
Decant water and replace with fresh water. The gel can be stored like this for days.
Silver Staining for Mass Spectrometric Analysis
(Use 5 gel volumes of each reagent for staining)
Step
Solution per gel
Time(min)
Fix
5% acetic acid; 45% methanol; 50% water
90
Wash
water
10
Wash
water
10
Sensitization
0.02% sodium thiosulfate
3
Wash
water
0.5
Wash
water
0.5
Silver
0.1% silver nitrate
30
Wash
water
0.5
Develop
0.02% formaldehyde/2.5% sodium carbonate
2
(bands suitable for MS analysis will appear within 30-60 seconds)
Stop
1% acetic acid
10
Wash
water
20
Wash
water
20
Alternate Version: Destaining Silver from the gels
WORKING SOLUTION
30 mM Potassium Ferricyanide
99 mg of Potassium Ferricyanide in 10 ml water
100 mM Sodium Thiosulfate
248 mg Sodium Thiosulfate in 10 ml water Mix at 1:1 ratio. Make fresh for use.
Cover the band in working solution and vortex until brown color removed (5 minutes) Remove the solution and wash three times with 200 ''L water.
In-gel Digestion
100 mM Ammonium Bicarbonate
0.79 g Ammonium Bicarbonate Make up to 100 mL with water
10 mM DTT
15.4 mg Dithiothreitol
Make up to 10 mL with 100 mM ammonium bicarbonate
55 mM Iodoacetamide
102 mg Iodoacetamide
Make up to 10 ml with 100 mM ammonium bicarbonate
0.1 g/l Trypsin
200 l of 25 mM Ammonium Bicarbonate
20 g trypsin
Excise gel bands prior to in-gel digestion
Digestion
Add enough volume of DTT to the gel pieces to cover.
Reduce for 30 min at 56 ''C
Cool to room temperature.
Replace DTT with iodoacetamide solution with occasional vortexing.
Alkylate for 30 min in dark with occasional vortexing.
Wash the gel pieces with 50-100 ''l ammonium bicarbonate, for 10 min.
Dehydrate with ACN. Vortex for 10 min. Discard the liquid.
Re-swell by addition of ammonium bicarbonate again. Vortex for 10 min
Shrink again with ACN. Vortex for 10 min. Discard liquid. Dry the gel pieces for 10 min.
Add 1-10 ''g of trypsin(according to the spot color)
Incubate for 18 hrs at 37 ''C.
Extraction (Note, this extraction protocol is not suitable for hydrophobic proteins)
Pool the digested liquid.
Extract with 25 mM ammonium bicarbonate. Pool it in the same tube.
Extract with 5% Formic Acid/50% Acetonitrile three times pooling all liquids.
Concentrate final product to 20 ''l.
Desalt
Use C18 Zip tips.
MALDI-MS
Make a saturated solution of 10 mg ''-cyano-4-hydroxy-cinnamic acid in 1 mL 60%ACN/0.1%TFA. Vortex and centrifuge. Use the supernatant only.
Mix the analyte and the matrix in the ratio of 3:1 ' 6:1 (v:v). Vortex. Use 1 ''l of this to spot on the target plate.
For calibration, mix 1 ''l of Angiotensin II and 1''l of ACTH. Add 6 ''l of matrix. Vortex. Spot 1 ''l onto the target plate.
Follow instrument instructions for obtaining mass spectra.
Database Searching
Once you have acquired your mass spectral data, you will need to search the experimental peptide masses against database values. The first step is to go to one of the database front-end sites. Any of the following are suitable and are listed only in alphabetical order:
Mascot
http://www.matrixscience.com/home.html
MOWSE
http://www.seqnet.dl.ac.uk/Bioinformatics/Webapp/mowse/
Peptide Search
http://www.mann.embl-heidelberg.de/Services/PeptideSearch/
Protein Prospector
http://prospector.ucsf.edu/
Prowl
http://prowl.rockefeller.edu/
After reaching the front-end interface, follow the guidelines for the particular site for entering your information. Below are some useful hints for database searching:
APPLICATIONS:
CHARACTERISATION OF PROTEINS:
Alternative protein separation techniques are available but 2-D gel electrophoresis is regarded as a well defined technique because it can separate complex protein mixtures into 1000 well separated molecules
2.POST TRANSLATIONAL MODIFICATIONS:
Post translational modification is an integral step in protein synthesis. A series of modified protens are involved in various signaling pathways . 2-D electrophoresis has extensive applications in detecting these changes as well as quantifying them.
3.MOLECULE-MOLECULE INTERACTIONS;
A majority of molecules (suvh as proteins, nucleic acid) are larger complexes that are often static/transitory form.this technique enables to study these interactions as it involves improved separations between molecules
CONCLUSION: WHATS THE FUTURE FOR 2-D ELECTROPHORESIS IN PROTEOMICS?
In the present scenario of proteomics, 2-D electrophoresis has two major drawbacks and 3 main merits. The two drawbacks are that its very low efficient in analysizing hydrophobic proteins and is highly sensitive to dynamic range and quantitative distribution . advantages include- robustness, parallelism and its unique ability to analyze proteins at a high resolution . when all these are taken into account, its obvious that 2-D electrophoresis will be delivering technical advancements when sample of limited range of protein expressions are taken into account.
However one of the important area where 2- d electrophoresis will prove to be extremely beneficial is in the study of modification landscapes wherein how protein modifications combine to modulate certain protein activities in a cell.
REFERENCES:
GEL ELECTROPHORESIS- PRINCIPLES AND BASICS, DR.SAMEL MAGDELHIN,ISBN 978-953-51-0458-2,INTECH PUBLICATION, 4TH APRIL 2012
METHODS IN MOLECULAR BIOLOGY, VOL 12; GEL ELECTROPHORESIS- BY M.BURMEISTER AND L.ULANOVSKY, COPYRIGHT @1992 , THE HUMANA PRESS Inc; TOTOWA,NJ.
O'FARELL,P.H HIGH RESOLUTION TWO DIMENSIONAL ELECTROPHORESIS OF PROTEINS J.BIOL.CHEM 250,4007-4021(1975)
RABILLOUD.T. et al IMPROVEMENT OF THE SOLUBILIZATION OF PROTEINS IN TWO DIMENSIONAL ELECTROPHORESIS WITH IMMOBILIZED PH GRADIENTS ELECTROPHORESIS 18, 307-316(1997)