Separation of Biomolecules
- 1. Separation of biomolecules by 1. NATIVE PAGE 2. 2D PAGE 3. AGAROSE GEL ELECTROPHORESIS
- 2. Gel Electrophoresis Gel electrophoresis is a technique commonly used in laboratories to separate charged molecules like, DNA, RNA, proteins according to their size. Charged molecule move through a gel when an electric current is passed across it. An electric current is applied across the gel so that one end of the gel has positive charge and other end of gel has negative charge. The movement of charged molecules is know as migration. Molecules migrate towards opposite charge. The gel consist of permeable matrix, a bit like a sieve thorough molecules travel when an electric current is passed across it. Small molecules migrate through the gel more quickly and therefore travel further than large fragments that migrate slowly therefore will travel shorter distance. As a result molecules are separated by size.
- 3. Agarose gel vs polyacrylamide gel Agarose 1. A complex Polysaccharide derived from sea weed. 2. Consist of many molecules. 3. Gels contain long chain of interlinked sugar to form a mesh work. 4. Important in sepaartion of Much larger DNA fragments. 5. Separate DNA about 50-20000 bp. 6. Have comparatively low resolving power. 7. Typical concentration 0.5 to 2%. 8. Non toxic and easy to handle. 9. Non uniform pore size. Polyacrylamide 1. Made by digestion of acrylonitrile by nitrile hydratase. 2. Consist of one large molecule. 3. Gels are made up of chemical crosslinking of acrylamide and Bis- acrylamide, producing a molecular sieve. 4. Important in separation of proteinsa as well as small nucleic acid such as oligonucleotide, miRNA, tRNA etc. 5. Separate DNA about 5-500 bp. 6. Have high resolving power. 7. Typical concentration 6-15%. 8. Contain a potent neurotoxin.
- 4. 1. NATIVE PAGE Native Polyacrylamide Gel electrophoresis (PAGE) used is for Studying the composition and structure of native proteins. Here native means proteins are in properly folded state, not denatured. In Native PAGE Proteins are prepared in a non- reducing non- denaturing Sample buffer, which maintains the protein secondary structure and native charge density. Separation is based upon charge, size, and shape of proteins. Proteins remain enzymatically active after separation. Mobility depends on Proteins charge and hydrodynamic size. Useful for separation or purification of mixture of proteins.
- 5. • Blue Native PAGE BN- PAGE is a native PAGE technique where coomassie brilliant blue dye provides the necessary charges to the protein complexes for the electrophoretic separation. It permits a high resolution Separation of multi-protein Complexes under native conditions. The electrophoretic mobility of a complex is determined by the negative charge of the bound Coomassie Dye and the size and Shape of complex .
- 6. • Clear Native PAGE CN-PAGE separates acidic water –soluble and membrane proteins in a Polyacrylamide gradient gel , and in many cases has lower resolution than BN-PAGE. It uses no charged dye so the electrophoretic mobility of proteins is related to intrinsic charge of proteins. The migration distance depends on the protein charge, it’s size and pore size of the gel. CN-PAGE is milder than BN-PAGE so it can retain labile supramolecular assemblies of membrane protein complex that are dissociated under the condition of BN-PAGE. CN-PAGE used as a very efficient microscale separation technique for FRET analyses.
- 7. • Quantative preparative native continuous PAGE QPNC-PAGE is a Bioanalytical, high resolution and highly accurate technique to separate proteins quantitatively by isoelectric point. This varient of gel electrophoresis is used by biologist to isolate active or native Metalloproteins in biological samples and to resolve properly and improperly folded metal cofactor containing proteins in complex protein mixture.
- 8. 2. 2D PAGE 2D PAGE is a powerful and widely used method for the analysis of complex protein mixtures Extracted from cells, tissues or other biological sample This technique separate proteins in two steps according to two independent properties 1) First –dimension is Isoelectric focusing (IEF) which seperates proteins according to their Isoelectric point (pI). 2) Second – dimension is SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) , which separates proteins according to their molecular weight(MW) This technique was first developed by O’Farrell and klose in 1975.
- 9. SAMPLE PREPARATION Must Select appropriate method to get selected proteins from cellular compartment of interest. Must break all non-covalent Protein-protein, protein-DNA, protein-lipid interactions, disrupts S-S bonds. Must remove substances that might interfere with separation process such as salts, polar detergents(SDS), lipids, polysaccharide, nucleic acids Must prevent proteolysis, accidental Phosphorylation, oxidation, cleavage, Deamidation. Must try to keep proteins soluble during both phases of electrophoresis process. Protein solubilization - 8M Urea (neutral Chaotrope) - 4% CHAPS (Zwitterionic detergent) - 5-20 mM DTT (to reduce Disulphide) - 2-20 mM tris base(for buffering) - Carrier ampholytes or IPG buffer (up to 2% v/v) to enhance protein solubility and reduce charge-charge interractions.
- 10. 1. Isoelectric focusing (IEF) First Dimension Isoeletric point(pI) : PH at which a protein has a neutral charge, loss or gain of protons in a PH gradient (In a PH below their pI proteins carry a net positive charge and in a PH above their pI, they carry a net negative charge. 1. In IEF , proteins are separated by electrophoresis in a PH gradient based on their isoelectric point. 2. A PH gradient is generated in gel and an electric field is applied across the gel . 3. At all PH other than isoelectric point , protein will be charged . 4. At it’s isoelectric point, since the protein molecule carry no net charge it accumulates or focuses into sharp band. 5. Proteins applied in the first dimension will move along the gel and will accumulate isoelectric point.
- 13. IMMOBILIZED PH GRADIENT AND IEF RUN Immobilized PH gradients are used for IEF because the fixed PH gradient remain stable over extended run times over high voltages. This technique has high resolution,great reproducibility and allow high protein load. Isoelectric focusing is run in the same solution that are used to extract or Solublize the proteins. The IPG strips with the protein sample must be rehydrated in the rehydration/sample buffer during which proteins samples are loaded into the strips. After the run in IEF cell , proteins Focus as band on the strip according to their isoelectric points.
- 14. 2nd dimension:SDS PAGE 1. SDS-PAGE is an electrophoretic method for Separating polypeptides according to their molecular weights. This technique is performed in polyacrylamide gels containing sodium dodecyl sulfate(SDS). 2. SDS is an anionic detergent which denatures the protein by breaking disulfide bonds, SDS masks the charge of the proteins themselves and form anionic complex have a roughly constant negative charge per unit mass. 3. Besides SDS a reducing agent such as Dithiothreitol (DTT) is also added to break any disulfide bonds present in proteins. 4. When proteins are treated with both SDS and a reducing agent, the degree of electrophoretic separation within a polyacrylamide gel depends largely on the molecular weight of the protein. 5. SDS linearizes the protein so that they may be separated strictly by molecular weight.
- 15. SDS Run The Equilibrated IPG strip is placed on the top of SDS-PAGE gel which is submerged in a suitable buffer and sealed in place with agarose gel. An electric current is applied across the gel , causing the negatively charged proteins to move out of the gel and migrate across the gel. the proteins separate according to their sizes and therefore by their molecular weight. It is common to run marker proteins of known molecular weight in a separate lane in the gel, in order to calibrate the gel and determine the weight of unknown proteins by comparing distance travelled relative to protein.
- 17. Visualization 1. After electrophoresis the gel is stained to visualize the separated proteins. 2. Commonly used stains are Coomassie brilliant blue, Sypro ruby or silver stain different protein will appear as distinct spot Within gel. 3. In silver stain, silver Colloid is applied to gel. Silver binds to cysteine groups within the protein. The silver is darkened by exposure to ultra-violet light. The amount of silver can be related to darkness, and therefore the amount of protein at a given location on the gel. 4. Silver staining 100X more sensitive than coomassie brilliant blue stain
- 18. Advantages of 2D PAGE 1. 2D-PAGE can accurately analyze thousands of protein in single run. 2. High- resolution:This technology resolves proteins according to both pI and molecular mass and enables the characterization of proteins with post-translational modifications that affect their charge state. 3. 2D-PAGE is also used to study differential expression of proteins between cell types. 4. The proteins can be separated in pure form from the resultant spots.
- 19. Disadvantages of 2D PAGE 1. This technique include a large amount of sample handling. 2. Limited reproducibility, and a smaller dynamic range than some other separation methods. 3. Difficulty in separation of hydrophobic proteins. 4. It is also not automated for high throughput analysis. 5. Certain proteins are difficult for 2D-PAGE to separate, including those that are in low abundance, acidic, basic, very large, very small.
- 20. Application of 2D-PAGE The protein can be separated in pure form from spots which can be quantified and also analyzed by MS (Mass spectrometry) Host cell protein analysis Quality control of proteins. Posttranslational modification studies. To study proteomics. Provides important information correlating the absence or presence of individual protein characteristics of specific clinical conditions.
- 21. 3.AGAROSE GEL ELECTROPHORESIS 1. Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of macromolecules such as DNA, RNA, or proteins in a matrix of agarose. 2. Agarose is a natural polymer extracted from seaweed that forms a gel matrix by hydrogen-bonding when heated in buffer and allowed to cool. 3. It is the most popular medium for the separation of moderate and large sized nucleic acid and have a wide range of separation.
- 22. Principle Of AGE Gel electrophoresis separate DNA fragments by size in solid support medium such as agarose gel. Sample DNA is Pippetted into the sample well, followed by the application of an electric current which causes the negatively charged DNA to migrate (electrophorse) towards the anodal, positive(+) end. The rate of migration is proportional to size, smaller the fragments move more quickly and wind up at the bottom of the gel. DNA is visualized by including in the gel an intercalating dye, Ethidium bromide , DNA fragments take up the dye as they migrate through the gel, illumination with ultraviolet light causes the intercalated dye to fluorescence. A larger fragment fluorescence more intensely.
- 23. REQUIREMENTS 1. An Electrophoresis Chamber and power supply. 2. Gel casting trays- the open ends of trays are closed with tape while gel is being cast. 3. Sample comb- around whIch medium is poured to form sample wells in the gel. 4. Electrophoresis Buffer – Usually Tris- acetate-EDTA(TAE) or Tris-borate- EDTA(TBE). 5. Loading buffer-Which containing dense(glycerol) to allow the sample to fall into the sample wall. 6. Staining- By ethidium bromide. 7. Transilluminator-(An ultraviolet light box) Which used to visualize stained DNA in gels.
- 24. Concentration of Agarose gel The percentage of agarose used depends upon the size of the fragments to be resolved. The concentration of agarose is reffered to as a percentage of agarose to volume of buffer (w/v) and agarose gels are normally in the range of 0.2%to 3%. The lower the concentration of agarose the faster the DNA fragments migrate. In general if the aim is to separate large DNA fragments a low concentration of agarose should be used, and if the aim is to separate small DNA fragments high concentration of agarose is recommended.
- 25. STEPS 1. To prepare gel, agarose powder is mixed with electrophoresis buffer to the desired concentration and heated in micowave oven to melt it. 2. Ethidium bromide is added to gel (final concentration 0.5 ug/ml) to facilitate visualization of DNA after electrophoresis. 3. After cooling the solution,It is poured into a casting tray containing sample comb and allowed to solidify at room temperature. 4. After the gel has solidified, the comb Is removed , taking care not to rip the bottom of the wells. 5. The gel in plastic tray is Inserted horizontally into the electrophoresis chamber and is covered with buffer. 6. Sample containing DNA mixed with loading buffer are then pippetted into the sample wells, the lid and power loads are placed on the approaches, and a current is applied. 7. The current flow can be confirmed by observing bubbles coming off the electrodes. 8. DNA will migrate towards the positive electrode. 9. The distance DNA has migrated in the gel can be judged by visually monitoring migration of tracking dyes.
- 27. Application of Agarose gel electrophoresis Estimation of the size of DNA. Analysis of PCR products e.g in molecular genetic diagnosis. Separation of restricted genomic DNA prior to southern analysis,or of RNA prior to Northern analysis. Agarose gel electrophoresis is commonly used to resolve circular DNA with different supercoiling topology, and to resolve fragments differ due to DNA synthesis. In addition to providing an excellent medium for fragment size analyses, agarose gels allow purification of DNA fragments.
- 28. Advantages of agarose gel electrophoresis 1. For most applications only a single-component agarose is needed and no Polymerization catalyst are required, therefore agarose gel are simple and rapid to prepare. 2. The gel is easily poured, doesn’t denature the samples. 3. The sample can also easily recovered.
- 29. Disadvantage of agarsoe gel electrophoresis The gels can melt during electrophoresis. The buffer can become exhausted. Different forms of genetic material may run in unpredictable forms.
- 30. Name:Md Amim Akram Faculty Roll no :18ZYB012 Enrollment No :GI0081 Bsc(Hons) zoology(mains) 5th Semester Thank you Prepared by