SDS PAGE.pptx sodium dodecyl sulfate PAGE

Sodium dodecyl sulfate PAGE

1. SDS PAGE AND ISOELECTRIC FOCUSING Submitted by Jasna C A 1 st MSc Microbiology MES MK Mackar Pillay College For Advanced Studies, Edathala

2. SDS PAGE SDS PAGE or Sodium dodecyl sulfate polyacrylamide gel electrophoresis, is a widely used technique in biochemistry and molecular biology. Used to separate proteins based on their size. Proteins with molecular masses between 5 to 250 kDa is separated. The combined use of sodium dodecyl sulfate and polyacrylamide gel eliminates the influence of structure and charge and thus proteins are separated by differences in their size.

3. PRINCIPLE SDS PAGE is a technique used to separate proteins based on their size. It relies on – vely charged SDS PAGE detergent, binds to protein, denatures them and imparts a uniform negative charge to each protein molecule. When an electric field is applied, proteins migrate through a polyacrylamide gel with smaller proteins moving faster than larger ones, leading to separation based on size. The separation is facilitated by the size of the pores within the gel ,with smaller proteins passing through more easily.

4. DENATURATION AND CHARGE Proteins in the sample are denatured and coated with SDS. This detergent binds to the proteins , denatures them and imparts a uniform negative charge due to its anionic nature. This ensures that the proteins mobility through the gel primarily depends on their size, not their charge. ELECTROPHORESIS An electric field is applied across the polyacrylamide gel. The negativity charged particles (proteins) migrate through the gel matrix towards the positively charged electrode . Smaller proteins move more quickly through gel because they encounter less resistance from the gel matrix.

5. INSTRUMENTATION GEL ELECTROPHORESIS APPARATUS This apparatus provides the platform for running the electrophoresis experiment. It typically consists of a rectangular tank or chamber with an upper buffer chamber and a lower buffer chamber separated by a vertical barrier called a comb or a spacer. The polyacrylamide gel is placed in this chamber. POWER SUPPLY The power supply is a critical component that provides the electrical potential required for the movement of charged proteins through the gel. It connects to the electrophoresis apparatus and generates a direct current (DC) electric field across the gel.

6. POLYACRYLAMIDE GEL The gel is a crucial part of the apparatus and serves as the separation medium. It consists of 2 distinct regions. The stacking gel The resolving gel The stacking gel has a lower acrylamide concentration and helps concentrate the protein sample into a narrow band before enter the resolving gel. This concentration steps enhances the resolution smaller proteins. The resolving gel has a higher acrylamide concentration, which affects the separation of proteins based on the proteins. SAMPLE LOADING WELLS Wells are created in the stacking gel for loading protein sample. These wells are typically formed by placing a comb or spacer in the gel solution during polymerization, creating cavities where the samples can be loaded.

7. MOLECULAR WEIGHT MARKER A molecular weight marker , also known as a protein ladder or protein marker, is a set of proteins with known sizes. It is loaded into a dedicated well on the gel alongside the protein sample. The molecular weight marker serves as a reference to estimate the size of the separated proteins based on their migration distances. STAINING AND VISUALIZATION EQUIPMENT After the electrophoresis run is complete, the gel needs to be stained to make the separated proteins band visible .
Various staining methods can be used , such as Coomassie Blue, Silver stain or fluorescent dyes.
The stained gel is then visualized using imaging equipment, which may include a gel documentation system or a simple gel scanner.

8. METHODS OF SDS-PAGE Materials and Reagents : Proteins sample Polyacrylamide gel components Tris -glycine SDS running buffer SDS-PAGE gel apparatus Molecular weight marker Sample buffer Loading buffer Staining solution Detaining solution Gel documentation system or scanner.

9. PROCEDURE 1. GEL PREPARATION Prepare a polyacrylamide gel with a stacking gel on top and a resolving gel below. The composition of the gel depends the expected size range of the proteins you are analysing. Typically, a 12% resolving gel is used for most applications. Polymerization the gel by adding ammonium persulfate and TEMED (Tetra Methyl Ethylene Diamine) to initiate polymerization. Insert a comb or spacer to create sample loading wells in the stacking gel. 2. SAMPLE PREPARATION Prepare your protein samples. If they are not already denatured, heat them at 95-100°C for a few minutes in the presence of SDS and a reducing agent . Mix the denatured samples with the loading buffer to ensure that they sink into the wells and have a consistent density.

10. 3. LOADING SAMPLES Load the prepared protein samples into wells of the stacking gel. Load a molecular weight marker into a dedicated for estimating protein sizes. 4. ELECTROPHORESIS Place the gel in electrophoresis apparatus and immerse it in the Triglycine SDS running buffer. Connect the apparatus to a power supply and apply a constant voltage (around 120-200V depending on gel size and setup). Allow the electrophoresis to run until the dye front reaches the bottom of the gel. This can take 30 minutes to a few hours, depending on the gel thickness and voltage. 5. VISUALIZATION After electrophoresis, carefully remove the gel from the apparatus. Stain the gel with a protein specific dye (e.g., Coomassie Blue) following the manufacturer instruction. If using Coomassie Blue, wash the gel with destaining solution to remove excess stain. The protein bands should now be visible in the gel.

11. 6. ANALYSIS Analyse the gel using a gel documentation system or scanner to capture an image of the separated proteins. Estimate the sizes of separated proteins by comparing their migration distances to the molecular weight marker lanes. APPLICATION Protein separation and analysis. Protein identification. Protein purification. Protein quality control. Research in forensic science. Research

12. ISOELECTRIC FOCUSING Isoelectric focusing (IEF) is a technique used to separate protein or molecules based on their isoelectric points ,the pH at which they carry no net electrical charge. Its commonly used in proteomics and research to gain insights into protein charge properties and purity. Materials and Reagents Isoelectric focusing gel. Protein sample Isoelectric focusing apparatus Electrodes Immobilized pH gradient buffer Sample buffer Staining solution Destaining solution Isoelectric point markers

13. PROCEDURE 1. GEL PREPARATION Prepare the isoelectric focusing gel, typically consists of an IPG strip or gel with a pH gradient ranging from acidic to basic. Rehydrate the IPG strip or gel in IPG buffer, ensuring it absorbs and rehydrates completely. 2. SAMPLE PREPARATION Prepare your protein sample in a buffer suitable for IEF. The buffers pH should be different from the expected pl of your proteins. Mix the sample gently to ensure they are well suspended. 3. LOADING SAMPLES Load the protein samples on the IPG strip or gel. This is typically done by placing the strip on the top of gel or by filling wells in the gel. Include pl markers to aid in determining the pl values of the separated proteins.

14. 4. ISOELECTRIC FOCUSING Assemble the IEF apparatus, ensuring the IPG strip or gel is properly positioned. Connect the electrodes to the apparatus and fill the apparatus with fresh IPG buffer to ensure conductivity. Apply a voltage across the IPG strip or gel. The proteins will migrate through the pH matching their pl. IEF may take several hours to complete, depending on the length of IPG strip or gel and the voltage applied. 5. VISUALIZATION After focusing, carefully remove the IPG strip or gel from the apparatus. Stain the proteins with a suitable staining solution to make them visible. If using Coomassie Blue, destain the gel to remove excess stain . 6. ANALYSIS Analyse the gel to observe the separated proteins and their relative positions along the pH gradient. Compare the positions of the pl markers to estimate the pl values of separated proteins.

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