Mol Bio Mid Review part 3

• separation of proteins by size and shape: The beads contain aqueous spaces into which small proteins can pass, slowing down their progress through the column. Larger proteins cannot enter the beads, allowing them to pass more rapidly through the column
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• usually is performed for purification of multi-protein complexes
• the same principal as antibody affinity chromatography but in this case, the beads are in a suspension instead of being in a column

• The cell extract is prepared under a gentle condition so the protein complexes remain intact (interacting proteins stay together).
• An antibody (bound to the beads) which is specific for the protein of interest is then added, which results in precipitation of this protein and all other proteins attached to it.
• Next, the beads are rinsed to remove all other unbound proteins.
• These attached proteins are separated and analyzed by different methodologies to identify one of the interacting proteins
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Proteins are separated by their molecular weights. To do this, the cell lysate is mixed with SDS and BME (Beta mercapto ethanol) and heated. Proteins are linearized (so shape would not be a factor) and made to have similar charges (all becoming negative charge, then variability of charge would not be a factor.

• BME opens up the disulfide bonds. SDS also helps to open the other bonds and surrounds the proteins & gives them negative charge. All proteins then become linear & become negatively charged.
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• sizes (molecular weights);
• Smaller; Larger ones

The gel may be stained by a protein dye to visualize the protein bands.

• After proteins are separated by electrophoresis, they are transferred to filter paper in a manner that retains the same relative position of the proteins. After blocking nonspecific protein binding sites, antibody binding is then detected using an attached enzyme (HRP) that creates light when it acts on its substrate.
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HRP; washes are done to remove the unattached antibodies

• The amino terminal is labeled and then 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. Determining 8-15 amino acids is sufficient for deciphering what protein is sequenced.
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• In the first dimension, the proteins are separated by isoelectric focusing. The strip of gel containing these protein bands is cut and soaked in SDS+BME by 90⁰ and a 2nd electrophoresis (in a denaturing gel) separates the proteins according to their sizes.
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• Protein samples include presence of hydrophobic and non-soluble proteins.
• low reproducibility
• difficult to analyze and compare 2 proteomes

label the constituents of 2 proteomes with different fluorescent colors (one sample all proteins green, the other same all proteins red) and run them together in a single 2D gel. Visualization of the 2D gel under different lights enables the intensity of equivalent spots to be compared (2 different colors for each spot, red from one sample and green from the other)

2D gel electrophoresis; excised from the gel and digested with trypsin to produce a series of peptides.

Most proteins are digested into a series of peptides of 5-75 amino acids in length.

• pulse of energy from a laser & then accelerated down the column to the reflector and onto the detector
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mass-to-charge ratio

• The data is visualized as a spectrum. By using a database of the predicted molecular masses, it is possible to determine the identity of each pick
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• identify what other proteins it interacts with and the consequences of those interactions (up-regulation, down-regulation, targeting for degredation);
• DNA cloning and DNA library

• fragment of DNA;
• cloning vector;
• propagation;
• host organism

• 1. recombining each DNA fragment into a cloning vector (plasmid)
• 2. propagating each vector-insert (the DNA fragment) in an organism (most of the time, E. coli)

Each colony of bacteria contains the cloned DNA fragment

• different cloned DNA fragments each one in one colony of bacteria. A library contains many colonies of bacteria;
• vector (plasmid);
• insert (DNA fragment)
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the viruses coat gene; one of the human proteins on their coats

• (A cDNA library represents different coded proteins in for a example, a human cell.)
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• protein of interest;
• phage display library (many phages, each has one type of human protein on its surface);
• those displaying a protein that interacts with the protein of interest
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The retained phages can be isolated and their chimeric "coat gene DNA - human cDNAs" can be sequenced

protein-protein interactions; binding between 2 proteins

• DNA binding domain & RNA polymerase binding domain
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• human protein A (protein of interest);a library of different human proteins (cDNA)
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• yeast DNA binding and RNA polymerase binding domains becoming close to each other. The activator therefore becomes functional to induce transcription of a reporter gene.
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protects the DNA from digestion by DNase; where on the DNA the protein is bound

ex. Lac repressor: a protein that binds to a particular sequence of the DNA (operator of Lac operon) can show the foot printing

• the collection of fragments generated by DNase cutting at sites in free DNA but not in DNA with repressor bound to it.
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• which feature of the DNA structure is necessary for the binding of a protein to it;
• chemically modifying the DNA (either at phosphate, sugar, or bases) and then mixing it with the protein of interest, performing foot printing assay

• disappear in the gel shift
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the fragment of the DNA that is occupied by the protein within the cell

• cross linked to DNA (stable chemical linking). This leads to DNA breaking down into small fragments
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• an antibody binding to the protein that is bound to the DNA;
• the protein to be removed, reversing the crosslinks. This amplifies & identifies the isolated DNA fragments
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