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Separation of Protein - Dialysis, Salt in, Out
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Protein separation, solubilization, fractionation, proteolytic inhibition, questions

Saranya v
Experienced Assistant professor with PH. D in biomaterials. coached for competitive exams. have set questions for competitive exams

Unacademy user
thank u mam for these videos 👍👍👍👍
Mam more video on biophysics pllz
hello mam..kindly let me know why we go for salting in and salting out method??
Saranya v
3 months ago
As different proteins have different compositions of amino acids, different protein molecules precipitate at different concentrations of salt solution. Unwanted proteins can be removed from a protein solution mixture by salting out as long as the solubility of the protein in various concentrations of salt solution is known. After removing the precipitate by filtration or centrifugation, the desired protein can be precipitated by altering the salt concentration to the level at which the desired protein becomes insoluble.
Divya Deogharia
3 months ago
okay mam..got it. thanku
Saranya v
3 months ago
welcome
  1. CSIR unit 13: Methods in Biology : A: Separation of protein


  2. Outline Plasmid Importance Steps of isolation Question discussion Steps Separation based on size Separation based on conformationn


  3. Steps of Protein separation 1. Solubilization 2. Prevention of protein aggregation 3. Reduction and denaturation of protein 4. Removal of contaminants Preparation of sample 1. Extraction of protein from the sample material 2. Protein solubilization ahead of analysis 3. Disruption of non-covalently bound proteins


  4. ample preparation steps Cell lysis Protection from proteolysis Fractionation Solubilization Removal of contaminant Quantification


  5. Cell lysis significance . To facilitate effective disruption of cells or tissues . To isolate proteins from intact cells and tissues while avoiding loss or modification of proteins To obtain all proteins in sample . Helps to maximize the sample recovery, retain structural integrity


  6. Steps . Cell disruption . Protection from proteolysis . Homogenization and sample solubilization


  7. Gentle lysis Employed when sample consists of easily lysed cells . E.g. tissue culture cells, blood cells Employed when particular sub-cellular fraction is to be analyzed Gentle lysis Freeze- thaw Osmotic Enzymatic Rapid freeze-thaw Suspend hypotonic medium cells inDetergent cycles Suspend cells in Cellulase plant cells detergent solution Lysozyme-bacteria


  8. Vigorous lysis . Employed when sample consists of cells which can't be easily lysed . E.g. Bacterial and plant cells Harsh lysis> Manual Sonication Homogenigrinding French Pressure zation (bead beater, ron


  9. Proteolysis ?? Cell lysis releases proteases which may result into proteolysis During the sample preparation protease inhibitors are added to minimize artifactual proteolysis


  10. Protease Inhibitor Effective protease inhibitors contains a mixture of irreversible and reversible protease inhibitors that inhibit serine, cysteine and metalloproteases . Specifically, useful for gel-based proteomics


  11. Effective against serine and Phenyl Methyl Sulphonyl Fluoride PMSF cysteine proteases Inactivated by DTT, unstable Ethylene Diamine Tetraacetic Acid Effective against metalloproteases Inhibits nucleases EDTA Ethylene Glycol Tetraacetic Acid Effective against metalloproteases EGTA . Sample processing in cold condition also reduce proteolysis


  12. Fractionation . Fractionation makes it possible to isolate groups of proteins, or fractions from total proteome Simplifies analysis of complex protein mixtures . Allows for improved resolution when an individual fraction is analyzed Provides less crowded 2-D maps


  13. alting Out precipitation Most proteins are less soluble at high salt concentrations, an effect called salting out. Salting In Proteins are usually poorly soluble in pure water. Their solubility increases as the ionic strength increases, because more and more of the well-hydrated inorganic ions are bound to the protein's surface, preventing aggregation of the molecules (salting in).


  14. -DIALYSIS BAG C CUT OFF SIZE BuFFER ( 1 NTER EST


  15. Questions the curious you


  16. Protein purification refers to the a) Purification of proteins b) Separation of proteins from other biomolecules c) Separation of a particular protein from other contaminating proteins d) all of these


  17. Protein separation techniques are often based on the following properties except a) solubility of the protein b) viscosity of the protein c) charge of protein d) specific binding affinity of the protein


  18. Which of the following detergent is commonly used to release integral proteins from its membranes? a) urea b) dimethyl sulphoxide c) triton X 100 d) cyanogen bromide