Flashcards
Protein Separation Part 1
Cards in this deck (23)
- 1AmphiphilicPositive and negative charges on the same molecule. Charges depend on the surrounding pH.
- 24 Properties of Proteins for Purification1. Solubility (function of salt, pH, temperature)
2. Charge
3. Size
4. Binding Properties (Ligands) - 3Salting out (Salt Precipitation)Removes the hydration shell around proteins at the salting point--where water is bound to the salts, and can no longer hydrate proteins. Ex. (NH4)2SO4
- 4Salting out (Salt Precipitation) Procedure and Problem1. Salt is added at a concentration (below to salting out).
2. Centrifugation then removes proteins that salted out or became less soluble.
3. Salt concentration is increased to allow salting out of desired protein.
4. Solution is centrifuged, and precipitate is collected that holds the desired protein.
Problem: Large salt conc contaminate solution and needs to be removed by dialysis. - 5Isoelectric PointWhen the pH results in the charge of the protein being 0. Proteins have different isoelectric points due to different AA seq.
- 6Isoelectric Precipitation and ProblemProtein aggregate at their pI, since there is no electrostatic repulsion keeping them apart. Trichloroacetic Acid can be used to adjust the pH of the solution.
Problem: Risk denaturing your proteins and thereby losing the activity of the protein. - 7Solvent FractionationDielectric constant of a solution alters the magnitude of the electrostatic interactions between charged groups. Decreasing the dielectric constant increases electrostatic interaction between charged species. This decreases the solubility of proteins as they become less ionized--electrostatic repulsion is not enough to prevent aggregation.
Adding water soluble organic solvents (ehtnaol/acetone) allows proteins to precipitate as the dielectric constant decreases. - 8Solvent Fractionation Optimum values5 to 60% of organic solvent is added to allow for precipitation. Performed at 0*C or below to prevent protein denaturation caused by temperature increases.
- 9Phenol Extraction and ProblemPhenol is added to separate DNA from proteins by utilizing the non-polar residues of the protein, that is exposed after mixing with the phenol (non-polar). Addition of water allows for the formation of a polar and non-polar phase, and your protein is separated.
Problem: Difficult to distinguish the two phases. - 10Denaturing Contaminated ProteinsDenaturing unwanted proteins by use of high temperatures or extreme pHs. Proteins stable at these extremes are then easily separated from the precipitated denatured proteins.
- 11Types of Chromatography (4)1. Gel Filtration/Size Exclusion: separates based on size (molecular weight)
2. Ion Exchange (cation/anion exchange): Separates by surface charge on proteins.
3. Hydrophobic Interaction: Separates by hydrophobicity of proteins
4. Affinity: Separates by unique binding characterisitc of protein of interest for affinity matrix in column. - 12Cation and Anion ExchangeCation: separates based on positive charges of solutes/proteins, matrix is negatively charged.
Anion: separates based on negative charges of solutes/proteins, matrix is positively charged. - 13Ion Exchange ChromatographyReversible adsorption-desorption of ions in solution to a charged solid matrix/polymer network. Buffer conditions (pH and ionic strength) are adjusted to favor max binding of the protein of interest. Contaminating proteins bind less strongly and pass through. Protein of interest is then eluted using another buffer solution which favors its desorption from the column.
1. Separation based on overall charge of moleucles.
2. Matrix retards passing proteins of opssite charge. Ex. DEAE cellulose (+)
CM-cellulose (-) - 14Ion Exchange Chromatography AND Salt GradientAllows for the separtion of proteins with different charges. Very high salt concentrations will allow for collection of proteins.
- 15Affinity ChromatographyNon-covalent binding of ligand to protein, similar to SPR. Ligand should be specific to protein.
- 16Possible Elution Strategies for Affinity Chromatography (4)1. pH
2. Ion Strength
3. Denature
4. Competitor ligand or analog - 17Key Stages in AC (4)1. Incubation of sample and binding of sample to ligand using binding buffer.
2. Adsorption of target and elution of unbound particles
3. Elution of sample by addition of high concentration of free ligand or change of conditions.
4. Requilibiration of the column
Problem: Protein bound to ligand, but now is at least free from column. - 18Common AC Columns1. Ni2+ binds to poly hisitidines (example 6xHis)
2. Specific Antibodies (anti-Flag tag)
3. Glutathione > binds to GST
4. Protein A or G binds antibodies - 19Specific Antibodies (anti-Flag tag)1. Fuse short peptide to desired protein.
Ex: HA, Myc, FLAG.
2. Purify with afinity beads that contain monoclonal AB that bind to the specific peptides.
3. Elute with peptide. - 20Glutathione binds to GST (Glutahione-S-Trasferase)
- 21Ni2+ binds to poly hisitidines (example 6xHis)Ni-NTA Columns: Ni-NTA resins bind to 6xHis tagged proteins/peptides. Highly specific interaction. Elution is done by reducing pH (acidic conditions), or competition by imidazole.
- Monomers eluted at pH~5.9 and concentrations greater than 100mM.
- Multimers elute at pH 4.5 and/or 200mM imidazole. - 22ImidazoleSimilar in structure to histidines which will compete with the tag to bind to the Ni atoms.
- 23Problem with Ni-NTA ColumnsProteins with neighboring histidines (non-tagged cellular proteins) can bind to Ni-NTA. True for eukaryotic cells, not bacteria.
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- Bioanalytical Chemistry Flashcards on Protein Separation Part 1, created by Aaysha Babaria on 26/10/2018.
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- Aaysha Babaria
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