Techniques of Molecular Bio part 1
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used for purification of multiprotein complexes
difference between antibody affinity chromatography & immunoprecipitation
In immunoprecipitation, beads are in a suspension instead of being in a column
In immunoprecipitation, the cell extract is prepared under a __
gentle condition so the protein complexes remain intact (interacting proteins stay together)
steps in immunoprecipitation
- A cell extract is prepared under a gentle condition. (cell lysis)
- The proteins are in a test tube (small circles).
- An antibody (orange) which is specific for the protein of interest is added and binds to the antigen of the protein complex.
- Beads (grey) are added to bind to the antibody which separates the proteins by masses.
- After centrifuging, the protein complex of interest precipitates to the bottom of the tube.
- The beads are rinsed to remove all other unbound proteins.
- The attached proteins are separated & analyzed by different methods to identify each one of the interacting proteins.
method of separating proteins by their sizes (molecular weights) using charge
PAGE (polyacrylamide gel electrophoresis)
To separate proteins by their molecular weights, proteins should be __
- linearized (so shape wouldn't affect the molecular weight) - done by BME opening up disulfide bonds
- all have similar charges (all negative charges) - SDS gives them this charge
During PAGE, the cell lysate is mixed with __
SDS & BME (beta mercapto ethanol)
purpose of BME
opens up disulfide bonds (acts as a reducing agent during PAGE)
purpose of SDS
acts as a denaturing gel during PAGE & helps open up other bonds. It surrounds the proteins & gives them negative charge
purpose of staining the gel with protein dye during PAGE
helps to visualize the protein bands (dye should not interact with the protein)
steps in PAGE
- Cell lysate is mixed with BME (to open disulfide bonds) & SDS (to break other bonds & give negative charge). Dye is also added to help visualize the proteins.
- Electrophoresis moves the protein (in the wells) from the anode to cathode. Smaller proteins will move faster.
- Proteins are now separated by molecular weights on the gel.
used to detect the protein of interest after electrophoresis
immunoblotting (Western Blotting)
steps in immunoblotting (Western Blotting)
- After proteins are separated by PAGE, they are transferred to a membrane with high affinity for the protein. The membrane is now a replica of the gel.
- Blocking: Protein solution like milk or BSA is added to cover all the areas of the membrane with no protein.
- The primary antibody is added which is specific to the protein of interest and will stick to the protein bands.
- A wash is done which removes the unattached antibodies.
- Then a secondary antibody is added that is specific to the primary antibody.
- The enzyme HRP allows the secondary antibody to recognize the primary antibody and bind to them.
- Another wash is done to remove the unattached antibodies.
- An enzyme is added which creates light (enhanced chemiluminesence) when it acts on its substrate. The light can be detected by different methods.
purpose of blocking in immunoblotting (Western blotting)
The membrane is sticky to all proteins including antibodies. Blocking covers all the areas of the membrane with no protein so that when antibodies are added, they will only stick to the proteins.
method in which a sample of purified protein is analyzed by sequencing a single protein
steps in Edman degradation
- The amino terminal is labeled.
- The first amino acid is removed without hydrolyzing the rest of the amino acids in the peptide.
- The released amino acid can be analyzed by HPLC.
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