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MALDI-TOF (matrix-assisted laser desorption ionization time-of flight) is used after __ in which a spot of protein is excised from the gel and __
2D gel electrophoresis; 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.
MALDI-TOF: 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 & consequences of such interactions (up regulation, down regulation, targeting for degradation, etc.);
- DNA cloning; cDNA 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)
Most eukaryotic mRNAs have a __ tail at their 3' end. A __ and __ can make a __, known as a cDNA
- poly-T primer;
- reverse transcriptase;
- complementary strand to the mRNA
To make a cDNA library, the __ component of RNA-DNA is degraded and the __ is made.
- second strand of cDNA
A cDNA library is the collection of __
all the cDNA clones generated from a particular isolated total mRNA from the cell
(Each cDNA is cloned by inserting into a vector and propagated in bacteria.)
It is possible to make a cDNA library in a __ instead of in plasmid & then infect bacteria with these constructs.
phage genome (vector)
- Bacteria that received (transformed by) these constructs generate the phages that carry the cloned cDNAs in their genome.
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 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.
yeast 2 hybrid assay: All yeasts are transfected with __ & each of them also is transfected with one of the constructs of __
- Hybrid 1; Hybrid 2
yeast hybrid 2 assay: If there is interaction between the human proteins, __
- there is gene expression & RNA polymerase is activated (If there is no interaction, there will by no gene expression.)
protein interaction map
- Each oval is a protein complex, with connections shown between complexes that share at least one protein. Each network is built up around a small number of proteins that have many interactions, and form hubs in the network, along with a much larger number of proteins with few individual connections.
the complete collection of metabolites present in a cell or tissue under a particular set of conditions
- the rate of flow of metabolites through the network of pathways that make up the cellular biochemistry
Changes are made to the genome by mutation or recombinant DNA techniques in order to influence the cellular biochemistry in a predetermined way.
EMSA (Electrophoretic Mobility Shift Assay) can be used to show __
interaction of a particular protein with a DNA fragment
(A protein is mixed with radiolabeled probe DNA containing a binding site for that protein. Acrylamide gel electrophoresis is used.)
EMSA: DNA not mixed with protein runs as a __ on the acrylamide gel. In the mixture with the protein, __
- single band corresponding to the size of the DNA fragment (left lane);
- a proportion of the DNA molecules (but not all of them at the concentrations used) binds protein molecules. Thus, there is a band corresponding to free DNA & another corresponding to the DNA fragment in complex with the protein
EMSA: __ before mixing it with DNA increases the size of the complex.
- Adding an antibody to the protein
- → slower movement → supershift
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