Sds page gel electrophoresis
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Introduction Gel (matrix) Polymerization of acrylamide Sodium dodecylsulphate Procedure
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Sds page gel electrophoresis
- 1. BY Sharad patange M pharm 1st year pharmacology
- 2. Introduction Gel (matrix) Polymerization of acrylamide Sodium dodecylsulphate Procedure
- 3. What is electrophoresis? Electrophoresis is a laboratory technique for separating molecules based on their charge.
- 4. SDS-PAGE ( sodium dodecylsulphate- polyacrylamide gel electrophoresis) The purpose of this method is to separate proteins according to their size, and no other physical feature And it is a step in Western blot
- 5. The gel (matrix) itself is composed of either agarose or polyacrylamide. Polyacrylamide is a cross-linked polymer of acrylamide. Acrylamide is a potent neurotoxin and should be handled with care!
- 6. Have smaller pores than agarose, therefore high degree of resolving power. Can separate DNA fragments which range in size from 10-500 bp. Polyacrylamide gel electrophoresis is also used to separate protein molecules.
- 8. Temed Cross-linked polyacrylamide gels are formed from the polymerisation of acrylamide monomer in the presence of smaller amounts of N,N’- methylenebisacrylamide (bis- acrylamide) Bisacrylamide is the most frequently used cross linking agent for polyacrylamide gels
- 9. SDS (sodium dodecyl sulfate) is a detergent that can dissolve hydrophobic molecules but also has a negative charge (sulfate) attached to it. If SDS is added to proteins, they will be soluablized by the detergent, plus all the proteins will be covered with many negative charges.
- 10. Now we are ready to focus on the second half - PAGE.
- 11. SDS Protein
- 12. So much SDS binds to proteins that the negative charge on the SDS drowns out any net charge on protein side chains In the presence of SDS all proteins have uniform shape and charge per unit length SDS nonpolar chains arrange themselves on proteins and destroy secondary tertiary and quarternary structrure
- 13. DC Power Source, Reservoir/Tank, Glass Plates, Spacers, and Combs Support medium Gel (Polyacrylamide) Buffer System High Buffer Capacity Molecules to be separated Proteins Nucleic Acids
- 14. Prepare polyacrylamide gels Add diluted samples to the sample buffer Heat to 95°C for 4 minutes Load the samples onto polyacrylamide gel Run at 200 volts for 30-40 minutes Stain
- 15. Mix ingredients GENTLY! in the order shown above, ensuring no air bubbles form. Pour into glass plate assembly CAREFULLY. Overlay gel with isopropanol to ensure a flat surface and to exclude air. Wash off isopropanol with water after gel has set (+15 min).
- 17. Tris buffer to provide appropriate pH SDS (sodium dodecyl sulphate) detergent to dissolve proteins and give them a negative charge Glycerol to make samples sink into wells Bromophenol Blue dye to visualize samples Heat to 95°C for 4 minutes
- 18. Run at 200 volts for 30-40 minutes Running Buffer, pH 8.3 Tris Base 12.0 g Glycine 57.6 g SDS 4.0 g distilled water to 4 liter
- 20. Chemical stains detect proteins based on differential binding of the stain by the protein molecules and the gel matrix. They are nonspecific in action, detecting proteins without regard to their individual identities. The important characteristics for a useful stain are: low background, high sensitivity, large linear range and ease of use.
- 21. smaller proteins will move through the gel faster while larger proteins move at a slower pace
- 22. Step by Step Instructions on how to assemble the polyacrylamide gel apparatus
Editor's Notes
- SDS-PAGE Denature protein(s) 100ºC, heat in presence of 1% SDS and 0.1M MCE This gives proteins uniform shape and constant q/m SDS binds denature protein 1.4g SDS/g Protein primarily through hydrophobic interactions with its long C12 tail, the SO4 head group decorates the protien with negative charges to give proteins constant q/m Thus, in an SDS-PAGE experiment electrophoretic mobility depends primarily on size, m. wt., since q/m and shape are essentially equal. ? Small molecules have a greater mobility than large molecules due to? A- sieving effect of the gel matrix ? Because A- all proteins under the same acceleration by E. Thus SDS-page is used to separate proteins by their molecular weight, identify proteins by their molecular weight or estimate an unknown protein’s molecular weight. The latter is done by plotting log MWT for some standard proteins of known molecular weight, run at the same time in the same gel , versus their relative mobility (*distance traveled into the gel relative to a dye front for instance). This technique works for proteins and protein subunits 10,000 – 200,000 mwt. Apparatus- vertical, vertical minis, tube and horizontal or flatbed design Two electrodes [anode (+) and cathode (-)] in conductive buffer: 2e- + 2H2O → 2OH- + H2↑H2O → 2H+ + ½ O2↑ + 2e- HA + OH- → A- + H2OH+ + A- → HA Apply equal and constant voltage on all cross-sectional areas of solid support. Electric field (volts/cm) Ohm’s law (E = IR) voltage is a function of current (I) and resistance (R) and since electrophoresis apparatus and buffers give a constant resistence, current is often used to define the voltage requirements ?What carries the current? A- Proteins and Nuclein Acids and all ions in solution Therefore we perform GE experiments in low ionic strenth buffer 0.05 – 0.15 M so thecurrent is not swamped with small molecule ions such that the proteins experimnce very little of the current and at pH 9 where most porteins have a negative charge and will migrate toward the anode
- (16 picograms = 16 × 10 -12 grams) in order to be detected. The gel has been stained with Coomassie brilliant blue.