Successfully reported this slideshow.
Your SlideShare is downloading. ×

final sds ppt.pptx

Loading in …3

Check these out next

1 of 56 Ad

More Related Content

Similar to final sds ppt.pptx (20)

Recently uploaded (20)


final sds ppt.pptx

  1. 1. SDS-PAGE
  2. 2. Presentors Areej Ahmed & Marryam Faiz Z19-24 & Z18-15
  3. 3. Jujges Muhammad Ibrahim Miraal Shehzad Hafsa Malhan
  4. 4. Contents: History Introduction Components and apparatus Principle Procedure Applications Advantages and disadvantages
  5. 5. History • In 1948, Arne Tiselius was awarded the Nobel Prize in Chemistry for the discovery of the principle of electrophoresis . • The discontinuous electrophoresis of 1964 by L. Ornstein and B. J. Davis made it possible to improve the separation by the stacking effect. • The denaturing effect of SDS in continuous polyacrylamide gels was first described in 1965 by David F. Summers in the working group of James E. Darnell to separate poliovirus proteins
  6. 6. …. • The SDS-PAGE was described in 1970 by Ulrich K. • But the technique was actually invented by Jake Maizel, who was doing a sabbatical in the MRC laboratory when Laemmli joined the lab as a postdoctoral fellow. • Maizel shared his prior technology with Laemmli and together they made further improvements. Laemmli and Maizel had planned to follow up with a Methods paper but this never materialized.
  7. 7. Introduction SDS PAGE is a technique used for the separation of proteins based on their molecular weight. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) SDS-PAGE is an electrophoresis method that allows protein separation by mass. Principle: I. The principle of SDS-PAGE states that a charged molecule migrates to the electrode with the opposite sign when placed in an electric field. II. The separation of the charged molecules depends upon the relative mobility of charged species. III. The smaller molecules migrate faster due to less resistance during electrophoresis.
  8. 8. Material required 1. Power supplies. 2. Gels. 3. Electrophoresis chambers. 4. Protein samples. 5. Running buffer. 6. Loading buffer. 7. Staining and destaining buffer. 8. Protein ladder. 9. Reagents
  9. 9. SDS 1. sodium dodecyl sulfate (SDS) is a popular anionic detergent for routine protein electrophoresis and cell lysis methods. 2. SDS is also present in the gel to make sure that once the proteins are linearized and their charges masked, they stay that way throughout the run. 3. This product is a powder ,for preparing SDS solution, dissolve the powder in H2O.
  10. 10. …. How to prepare SDS solution: 1. Weigh out 10 g SDS and add to a 100 mL Duran bottle. Be careful ,avoid inhalation and use a fu me mask. 2. Measure out 80 mL of distilled water and add to the Duran bottle. 3. Place on a magnetic stirring plate to mix the solution. 4. To dissolve SDS quicker, you can gently heat up the solution (60oC) using a hot plate or water ba th. 5. Once the powder has fully dissolved, top up the solution to 100 mL using distilled water. 6. An optional step is to filter the solution to remove any particles that have failed to dissolve.
  11. 11. ….
  12. 12. …. 1. The function of SDS is to break the hydrogen bonds of proteins 2. Masks the charge on protein so that all protein will act as negatively charged. 3. so that separation of the proteins is solel y based on molecular weight. 4. It is a denaturing reagent.
  13. 13. Beta mercaptoethanol 1. Beta-mercaptoethanol (BME) is a red ucing agent that acts on disulfide bonds. 2. In the absence of BME, proteins with disulfide bonds retain some of their structure 3. BME 4. BME is a poison if ingested, causes irrit ation in the mucous membranes, and can be absorbed dermally.
  14. 14. ….
  15. 15. ….
  16. 16. PAGE Polyacrylamide gel electrophoresis. Principle: when a charged molecule is exposed to an electric field, it migrates towards the electrode carrying the opposite charge. Function: PAGE is a biochemical technique that allows for proteins to be separated by their electrophoretic mobility . The gel is prepared by polymerizing acrylamide with the cross-linking agent N,N′-methylenebisacrylamide (bis-acrylamide) with the addition of ammonium persulfate .
  17. 17. …. Polyacrylamide: PAM is highly water-absorbent, forming a soft gel when hydrated Acrylamide : a colorless crystalline solid which readily forms water-soluble polymers. They form linear polymers Bisacrylamide :acts as the cross-linking agent, creating a network of polyacrylamide instead of linear polymers. The 'pore size' is determined by the ratio of acrylamide to Bisacrylamide. Ammonium persulfate: Catalyses the polymerization of acrylamides and poly acrylamides. TEMED: is used with ammonium persulfate (APS) to catalyze acrylamide polymerization when pr eparing gels for electrophoresis. Thermo Scientific Pierce Tetramethylethylenediamine.
  18. 18. ….
  19. 19. Gel In SDS-PAGE, the gel is cast in a buffer containing sodium dodecyl sulfate (SDS), an anionic detergent. 1. Stacking gel: The purpose of the stacking gel is to concentrate all of the different sized proteins into a compact horizontal zone by sandwiching them between a gradient of glycine molecules above and chloride ions be ow. It has lower amount of polyacrylamides. 2. Running gel: Based on their size and charge, the molecules will travel through the gel in different directions or at different speeds, allowing them to be separated from one another. It has higher amount of polyacrylamide.
  20. 20. ….
  21. 21. Electrophoresis: Electro: charge Phorsesis :carry “It is the movement of charged particles in a fluid or gel under the influence of an electric field.”
  22. 22. Loading buffer Loading buffers are reagents added to protein or DNA samples for loading into gels for electrophoresis applications. Loading buffers contain a colored dye, most commonly blue or orange, to visualize and track the progression of the bands across the gel. It contains: 1. 10% SDS 2. B-mercaptoethanol 10% 3. 20 % Glycerol 4. 0.12M Tris-HCL 5. 0.004% bromophenol blue dye 6. Sample
  23. 23. Running buffer The running buffer contains ions that conduct current through the gel. Tris-HCl 25 mM Glycine 200 mM SDS 0.1% (w/v)
  24. 24. Staining and destaining Once proteins are separated by gel electrophoresis, staining can be used to visualize the proteins. The most common method is staining with Coomassie blue, which after washing gives blue bands on a clear bac kground. Destain Solution is a uniquely formulated, ready-to-use reagent specifically designed for in-gel visualization of Coomassie Blue stained total protein in polyacrylamide gels. Continue the destaining until the protein bands are seen without background staining of the gel. Destain the gel by soaking for at least 2 hours 10% acetic acid. 50% methanol. 40% H2O . If the gel still has a Coomassie Blue background then continue destaining until the background is nearly clear
  25. 25. ….
  26. 26. Apparatus Voltage :200v Current:100mA to 120 mA
  27. 27. • se-gel-electrophoresis#:~:text=Proteins%20are%20generally%20smaller%20than,(sieve) %20size%20than%20agarose. • el-electrophoresis-of-DNA-is-horizontal • :text=SDS%2DPAGE%20allows%20an%20estimation,the%20standard%20or%20protein %20ladder. • • • • References
  28. 28. THANK YOU
  29. 29. Marryam Faiz
  30. 30. Contents: History Introduction Components and apparatus Principle Procedure Applications Advantages and disadvantages
  31. 31. Why Agarose is not used instead of polyacrylamide? • Proteins are generally smaller than DNA. • Thus, while DNA (larger than 100 bp) is routinely separated on agarose gels, proteins are generally run on polyacrylamide gels, as polyacrylamide matrices have a smaller pore (sieve) size than agarose. • Polyacrylamide is inert.
  32. 32. Principle • A charged molecule migrates to the electrode with the opposite sign when placed in an electric field. The separation of the charged molecules depends upon the relative mobility of charged species. V=qE/f • The smaller molecules migrate faster due to less resistance during electrophoresis. • Sodium dodecyl sulphate and polyacrylamide eliminate the influence of structure and charge of the proteins, and the proteins are separated based on the length of the polypeptide chain.
  33. 33. Procedure Gel Preparation 01 Sample Preparation 02 Electrophoresis 03 Gel Staining 04 Destaining 05
  34. 34. 1- Gel Preparation 1. Clean the glass plates and spacers of the gel casting unit with deionized water and ethanol. 2. Assemble the plates with the spacers on a stable, even surface. 3. Two types of gel are prepared • Separating gel • Stacking gel
  35. 35. 1. Prepare resolving gel solution using the following volumes (for 10 mL) depending on the percentage of gel required. Gel % Water (mL) 30% acrylami de (mL) 1.5 M Tris-HCl , pH 8.8 (mL) 10% SDS (µ L) 10% APS (µ L) TEMED*(µL) 8% 4.6 2.6 2.6 100 100 10 10% 3.8 3.4 2.6 100 100 10 12% 3.2 4.0 2.6 100 100 10 15% 2.2 5.0 2.6 100 100 10 *TEMED must be the last ingredient added 1- Separating Gel 2. Pour the gel solution in the plates assembled with spacers. To maintain an even and horizontal resolving gel surface, overlay the surface with water or isopropanol 3. Allow the gel to set for about 20-30 min at room temperature.
  36. 36. 1- Separating Gel 1. Pour acrylamide solution for a separating gel. 2. Overlay with water to prevent contact with air (oxygen), which inhibits polymerization. 3. Allow acrylamide to polymerize for 20-30 minutes to form a gel. Remove the overlaid water.
  37. 37. • Proteins migrate at different rate depending on the concentration of the separating gel. • Use an appropriate gel concentration for your target protein. • Using a higher acrylamide concentration produces a gel with a smaller mesh size suitable for the separation of small proteins. In general, an acrylamide concentration between 6 a nd 15% is used. • Gels with an acrylamide concentration gradient (gradient gels) are also used.
  38. 38. 1. Prepare stacking gel solution. 2- Stacking Gel Gel % Water ( mL) 30% ac rylamid e (mL) 0.5 M T ris-HCl, pH 6.8 ( mL) 10% S DS (µL) 10% AP S (µL) TEMED *(µL) 5% 5.86 1.34 2.6 100 100 10 *TEMED must be the last ingredient added
  39. 39. 1. Pour acrylamide solution for a stacking gel until it overflows. 2. Insert a comb and allow the acrylamide to polymerize ensuring no air bubbles are trapped in the gel or near the wells. 3. Allow the gel to set for about 20-30 min at room temperature. 2- Stacking Gel
  40. 40. • Proteins are highly concentrated when they migrate through a stacking gel prior to entering a separating gel. • The concentration occurs due to the difference in the migration rate of glycine ion, chloride ion, and proteins. 2- Stacking Gel
  41. 41. 2- Sample Preparation 1. Add sample buffer to samples (equal volume) and mix by flicking the tube. 2. Heat the samples at 100°C for 3 minutes in a heat block. 3. Centrifuge at 15,000 rpm for 1 minute at 4°C, and use the supernatant for SDS-PAGE.
  42. 42. 3- Electrophoresis 1. Remove the binder clips, spacer, and comb from the gel assembly, and mount the gel in the electrophoresis apparatus using binder clips. 2. Pour running buffer into the upper and lower chambers of the electrophoresis apparatus and remove air bubbles and small pieces of gel from the wells and under the gel using a syringe.
  43. 43. 3-Electrophoresis 3. Load samples and molecular weight markers in wells.
  44. 44. 3-Electrophoresis 4. Turn on the power supply, and run the gel until the dye (BPB) in the sample buffer reaches the bottom of the gel.
  45. 45. 3-Electrophoresis 5. Remove the gel assembly from the electrophoresis apparatus. Remove the gel from the glass plates using a spatula, and prepare for subsequent analysis.
  46. 46. 4- Gel Staining 1. Coomassie Blue staining: Staining of protein gels with Coomassie Brilliant Blue is a common procedure to visualize proteins resolved by SDS-PAGE. 2. Prepare Coomassie blue solution Coomassie Stain Solution: (1 l) Coomassie Blue 2 g Methanol 450 ml dH2O 450 ml Glacial acetic acid 100 ml 3. Place resolving gel in Coomassie Stain with gentle agitation for 30 min (note: the gel will shrink).
  47. 47. 5- Destaining 1. Destaining solution is prepared. Methanol 50 ml Glacial acetic acid 70 ml dH2O …to 1 l 2. The gel is soaked in the destaining solution for atleast 2 hours. 3. Destain may need replacing several times. To speed up destain, place a tissue in the tray with gel to soak up coomassie.
  48. 48. Apparatus Voltage :200v Current:100mA to 120 mA
  49. 49. Why the apparatus is kept vertically instead of horizontally like that in agarose gel electrophoresis?
  50. 50. Why is the apparatus kept vertically instead of horizontally like that in agarose gel electrophoresis? • They comprise a stacking gel and a resolving gel. A vertical arrangement allows you to make them sequentially. You pour the resolving gel first, and then once it is set, pour the stacking gel on top of it. This results in one contiguous gel. • The oxygen in air reacts with free radicals and inhibits the polymerization reaction. Therefore, this reaction would not proceed efficiently in an open, horizontal system. • Polyacrylamide gel is very thin compared to agarose gel. In horizontal position you coul d only load tiny amounts of the sample whereas at least 5 ml is required in each well.
  51. 51. Applications 1. It is used to measure the molecular weight of the molecules. 2. It is used to estimate the size of the protein 3. Used in peptide mapping 4. Estimate the purity of proteins. 5. Used in HIV test to separate HIV proteins. 6. To analyze post-translational modifications
  52. 52. Advantages and Disadvantages Advantages • Cross-linked gel is chemically stable. • Seperation can be done solely on size and weight. • Inexpensive • Good reproducibility Disadvantages • Polyacrylamide and SDS are both toxic and hazardous, they must be handled carefully. • Gel preparation is difficult and requires longer time. • Deliberate denaturation of proteins prior to electrophoresis (enzymatic activity, protein binding interactions generally cannot be determined by SDS-PAGE)
  53. 53. • se-gel-electrophoresis#:~:text=Proteins%20are%20generally%20smaller%20than,(sieve) %20size%20than%20agarose. • el-electrophoresis-of-DNA-is-horizontal • :text=SDS%2DPAGE%20allows%20an%20estimation,the%20standard%20or%20protein %20ladder. • • • • References
  54. 54. Any Questions?
  55. 55. THANK YOU