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gel filtration chromatography
- 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
- 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
steps of immunoprecipitation
- 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
polyacrylamide gel electrophoresis (PAGE)
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.
purpose of BME (Beta mercapto ethanol) & SDS
- 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.
Electrophoresis of the protein (after mixed with SDS & BME) on polyacriamide gel separates the protein according to their __. __ proteins move faster. __ move slower in the gel.
- sizes (molecular weights);
- Smaller; Larger ones
The gel may be stained by a protein dye to visualize the protein bands.
immunoblotting (Western blotting)
- 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.
The enzyme __ could have been linked to the detection antibody (primary antibody) or alternatively linked to a 2nd antibody that can interact with the primary antibody. Before adding the substrate enzyme, __
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.
2D gel electrophoresis
- 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.
problems with 2D gel electrophoresis
- Protein samples include presence of hydrophobic and non-soluble proteins.
- low reproducibility
- difficult to analyze and compare 2 proteomes
To compare 2 samples with 2D gel electrophoresis, it is possible to __
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)
MALDI-TOF (matrix-assisted laser desorption ionization time-of flight) is used after __ in which a spot of protein is __
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.
MALDI-TOF: In the mass spectrometer, the peptides are ionized by a __
- pulse of energy from a laser & then accelerated down the column to the reflector and onto the detector
MALDI-TOF: The time-of-flight of each peptide depends on its __
- 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
To determine the function of a protein, it is important to __. For better understanding of some of these protein methods, it is necessary to know about __
- 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
DNA cloning: insertion of a __ into a __ and subsequent __ of the recombinant DNA in a __
- fragment of DNA;
- cloning vector;
- host organism
DNA cloning involves the following steps:
- 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
A DNA library is a collection of __. The __ is the same in all, but the __ is mostly unique for each colony.
- different cloned DNA fragments each one in one colony of bacteria. A library contains many colonies of bacteria;
- vector (plasmid);
- insert (DNA fragment)
producing phage display library: A cDNA library is made into phage vectors in fusion with __ and then infecting bacteria with these constructs. Each phage particle produced by these transformed bacteria therefore display__
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.)
phage display method: The __ is immobilized within wells of a microtiter tray and the __ is added to the wells. After washing, the phages that are retained in the well are __
- 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
phage display method: After washing, how do we identify what protein was interacting with the protein of interest?
The retained phages can be isolated and their chimeric "coat gene DNA - human cDNAs" can be sequenced
The yeast 2 hybrid assay is used to discover __ by testing for __
protein-protein interactions; binding between 2 proteins
yeast 2 hybrid assay: The transcription factor is split into __
- DNA binding domain & RNA polymerase binding domain
yeast 2 hybrid assay: The DNA binding domain can activate transcription by binding to __. The RNA polymerase binding domain binds to __
- human protein A (protein of interest);a library of different human proteins (cDNA)
yeast 2 hybrid assay: Interaction of human protein A & B causes __
- 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.
nuclease protection foot printing: Binding of DNA to a protein __. This can be used to determine __
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
nuclease protection foot printing: DNA molecules are first bound to repressor and then subjected to DNase treatment. The "footprint" is indicated on the right (see photo). This corresponds to __
- the collection of fragments generated by DNase cutting at sites in free DNA but not in DNA with repressor bound to it.
The chemical interference foot printing method can help to identify __ by __
- 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
chemical interference foot printing: If modification of some bases causes lack of binding of the protein to the DNA the footprint will __
- disappear in the gel shift
ChIP (chromatin immunoprecipitation) assay can identify __
the fragment of the DNA that is occupied by the protein within the cell
ChIP assay: Proteins that are bound to DNA are __
- cross linked to DNA (stable chemical linking). This leads to DNA breaking down into small fragments
ChIP assay: Immunoprecipitation of the DNA fragments that are cross linked to the protein of interest is done by __, which causes __
- 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