Essentials of Gel Electrophoresis

This lecture is about Gel Electrophoresis and a little brief about it, which is presented by Tuba Nafees she is MSc graduate in Biotechnology from University of Karachi, Sindh Pakistan.
For youtube :https://www.youtube.com/watch?v=G4dwvDkxKN4

1. ESSENTIALS OF GEL ELECTROPHORESIS Presenter TUBA NAFEES M.Sc. In Biotechnology, University of Karachi 1

2. GEL ELECTROPHORESIS PRINCIPLE Biomolecules are separated and visualized on a gel as they migrate through it in the presence of an electric field. E= F/Q 2

3. TYPES OF GEL It is a purified form of agar ( Agar--- agarose+ agaropectin) Agarose is a linear polymer repeating units of Agarobiose ( D-galactose and 3,6-anhydro-L-galactopyranose). POLYACRYLAMIDE It is a polymer formed from acrylamide subunits, crosslinked to N,N'methylenebisacrylamide . AGAROSE ACRYLAMIDE BISACRYLAMIDE D-GALACTOSE 3,6-anhydro-L-galactose POLYACRYLAMIDE AGAROSE 3

4. AGAROSE Runs horizontally. Sets as it cools. Porosity is dictated by agarose concentration ( 0.5-2 %). DNA fragments of 20-20,000 bp in size Gel is thick. Low resolution ( mostly used for DNA separation). ( DNA single base pair = 650 Da) POLYACRYLAMIDE Runs vertically. Sets by a chemical reaction once crosslinking occurs. Porosity is dictated by acryl amide concentration DNA fragments of 5-500 bp in size & separate proteins of 5-200 kDa . Gel is thin. High resolution ( Protein separation) ( 1 Amino acid= 100 Da) 4

5. GEL CONDITIONS DENATURING GEL Biomolecule's natural structure is disturbed Nucleic acid by Urea RNA by DMSO & glyoxal Proteins by SDS Reducing conditions by beta- mercaptoethanol or dithiothreitol in SDS-PAGE Biomolecules are separated on the basis of charge to mass ratio. NATIVE GEL Biomolecule's natural structure is maintained No denaturing agent is used Biomolecules are separated on the basis of size, charge and shape . 5

6. allowing for analysis of only primary structure When to use? Blotting DNA & Protein sequencing allowing for analysis of all four levels of the biomolecular structure State of the sample Study of enzymes 6

7. FACTORS EFFECTING GEL ELECTROPHORESIS Electric Field Sample Buffer Supporting Medium Voltage. ( V= IR) Current Charge, size and shape Rate of migration α 1/ size and shape pH, migration of compounds Inert, Viscosity and Pore size. 7

8. GEL ELECTROPHORESIS APPARATUS GEL TANK POWERSUPPLY COMBS GEL CASTING TRAY RUBBER END CAP NEGATIVE ELECTRODE POSITIVE ELECTRODE GLASS SLIDES TANK LID 8

9. AGAROSE GEL ELECTROPHORESIS FOR THE SEPRATION OF DNA FRAGMENTS 9

10. Composition: Tris base + acetic acid+ EDTA Large pieces of DNA Low voltages, i.e. <150V Lower buffering capacity Low cost Composition: Tris base+ boric acid + EDTA. Small DNA fragments (0.1to 3kb). High voltages, i.e. 2000V. Higher buffering capacity High cost TAE BUFFER TBE BUFFER RUNNING BUFFER REAGENTS & CHEMICALS 10

11. GEL TRACKING DYES Bromophenol blue and Xylene Cyanol . Density ( Sucrose or glycero l ) DNA STAINING DYE Ethidium Bromide EtBr Orange G Bromophenol Blue Cresol Red Xylene Cyanol Agarose ( 0.5- 2%) Mix agarose powder with buffer (TAE/ TBE) in a flask and heat it. Cool down at 50 °C. Add EtBr to a final concentration of sol 11

12. A - Insert the tray into the groves of casting dam B -Insert the comb into the required position within the tray. Prepare the agarose gel and pour into the tray C -Once the gel becomes opaque carefully removes the casting dams before placing the gel within the tank. Gently Remove the combs. D -Pour buffer ( to cover the gel and fill the wells E -Comb may be inserted upside down and reinserted into the tray to provide convenient loading support. Red tape on underside of the tray or on the gel tank platform helps well detection. F -Remove the comb and place the lid. Pictures credit- Cleaver Scientific Limited. A D C B F E 12

13. DNA MARKER Sample DNA BANDS Smaller fragment Larger fragment Lanes DNA VISUALIZATION ON TRANSILLUMINATOR 13

14. PROTEIN SEPARATION BY SDS-PAGE 14

15. SDS is an anionic detergent that denatures secondary and non–disulphide linked tertiary structures, and additionally applies a negative charge to each protein in proportion to its mass. 15

16. REAGENTS & CHEMICALS Acrylamide Methylene bis acrylamide Tris HCL Sodium dodecyl sulfate ( SDS) Ammonium peroxide sulfate ( APS) TEMED ( tetramethylethylenediamine ) Glycine 2-mercaptoethanol Bromophenol Blue Coomassie Brilliant Blue 16

17. PREPARATION OF SOLUTIONS Acrylamide-bis- acrylamide Sol ( 30:0.8) Resolving Gel Buffer ( Tris HCL , pH :8.8) Stacking Gel Buffer ( Tris HCL , pH :6.8) SDS Solution ( 1 %) APS Solution ( 1.5 %) Reservoir Buffer ( Tris + Glycine+ SDS , pH :8.3) Sample Diluting Buffer ( Tris HCl+ SDS+ 2- mercaptoethanol + glycerol or sucrose+ Bromophenol blue crystals) Staining Solution ( Coomassie blue +Acetic acid + Methanol) Destaining Solution ( Acetic acid + Methanol) Dissolve protein in sample diluting buffer and heat it in boiling water bath for 3-4 mins. All the solutions are prepared using distilled water. 17

18. STACKING & RESOLVING OF PROTEINS RESOLVING GEL STACKING GEL 1 2 3 Glycine ( N/-) Proteins ( -) Cl - Cl - Glycine ( N/-) Proteins ( -) pH: 6.8 pH: 8.8 18

19. PROCEDURE A - Gather all the materials. B - Assemble the gel casting mold C - Pour acrylamide solution. Overlay with water Allow it to polymerize for 20-30 minutes .Remove the overlaid water. D - Pour acrylamide-bis-acrylamide sol for stacking gel and insert a comb. Allow it to polymerize D A B C OVERLAID WATER 19

20. E - Remove the comb , spacer and the binder clip. Place the gel in vertical assembly and fill it with running buffer F- Remove bubbles by using syringe G- Load sample into the wells H - Turn on the power supply and run the gel. H E G F 20

21. VISUALIZATION OF PROTEIN BANDS STAINING SOLUTION DESTAINING SOLUTION PROTEIN BANDS 21

22. APPLICATIONS OF GEL ELECTROPHORESIS PCR DNA FINGERPRINTING DNA SEQUENCING PROTEOMICS BLOTTING EVOLUTIONARY RELATIONSHIP 22

23. FOR ANY QUERY YOU CAN LEAVE YOUR QUESTION IN THE COMMENT SECTION 23

24. REFERENCES Smisek , D. L., Hoagland, D. A. (1989). "Agarose gel electrophoresis of high molecular weight, synthetic polyelectrolytes". Macromolecules . 22 (5), 2270–2277. Reece, J. B., Taylor, M. R., Simon, E. J., and Dickey, J. L. (2012). Gel electrophoresis sorts DNA molecules by size. In Campbell biology: Concepts & connections (7th ed., p. 243). Sambrook J, Russel DW (2001). Molecular Cloning: A Laboratory Manual 3rd Ed. Cold Spring Harbor Laboratory Press. Cold Spring Harbor , NY.  Kratz, R. F. and Siegfried, D. R. (2010). Using gel electrophoresis to separate molecules. In Biology for dummies (2nd ed., pp. 132-133). Hoboken, NJ: John Wiley & Sons. Oswald, N. (2008, August 6). Lopachin . R. (2004). "The changing view of acrylamide neurotoxicity". Neurotoxicology . 25 (4): 617–30. The principle and method of polyacrylamide gel electrophoresis (SDS-PAGE). Retrieved from https://ruo.mbl.co.jp/bio/e/support/method/sds-page.html 24

25. Fitzpatrick, Richard. (2007) “Electric Fields.” The University of Texas at Austin. How To Cast And Run An Agarose Gel in  The Multi Sub Mini Electrophoresis System Retrieved from https://youtu.be/zXgM10ghY_w How to stain an  SDS- PAGE Gel. Retrieved from https://youtu.be/b-1dXzU4iOw . http://www.cpet.ufl.edu/wp-content/uploads/2013/10/Intro-Gel- Electrophoresis-manual.pdf https://webfiles.uci.edu/treseder/public/Protocols/Agarose%20Gel%20Electrophoresis.pdf https://openwetware.org/wiki/Agarose_gel_loading_buffer   25

26. PICTURES CREDIT https://www.enasco.com/p/Edvotek-M12-Dual-Gel-Electrophoresis-Apparatus%2BSB45606 https://www.fishersci.co.uk/shop/products/tv100-standard-cooled-twin-plate-mini-gel-electrophoresis-unit/15805551 https://www.mikeblaber.org/oldwine/BCH4053l/Lecture05/Lecture05.htm https://www.google.com/url?sa=i&url=https%3A%2F%2Fgeneticeducation.co.in%2Frole-of-etbr-in-agarose-gel-electrophoresis-karyotyping%2F&psig=AOvVaw0LTwrmtAPixWpKyLQFU-AG&ust=1596730659749000&source=images&cd=vfe&ved= https://openwetware.org/wiki/Xylene_cyanol https://molbio.mgh.harvard.edu/szostakweb/protocols/protein_page/index.html https://en.wikipedia.org/wiki/Polyacrylamide https://ruo.mbl.co.jp/bio/e/support/method/sds-page.html http://www.protocol-online.org/biology-forums-2/posts/17700.html https://askabiologist.asu.edu/agarose-gel-electrophoresis https://videorista.com/index.php?route=product/product&product_id=62 https://en.wikipedia.org/wiki/Protein 26