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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.
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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
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MALDI-TOF: The time-of-flight of each peptide depends on its __
mass-to-charge ratio
- The data is visualized as a spectrum.
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MALDI-TOF: By using a database of the predicted molecular masses, it is possible to determine __
the identity of each pick
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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
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DNA cloning: insertion of a __ into a __ and subsequent __ of the recombinant DNA in a __
- fragment of DNA;
- cloning vector;
- propagation;
- host organism
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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
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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)
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Most eukaryotic mRNAs have a __ tail at their 3' end. A __ and __ can make a __, known as a cDNA
- poly(A);
- poly-T primer;
- reverse transcriptase;
- complementary strand to the mRNA
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To make a cDNA library, the __ component of RNA-DNA is degraded and the __ is made.
- RNA;
- second strand of cDNA
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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.)
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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.
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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.)
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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
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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
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The yeast 2 hybrid assay used to discover __ by testing for __
protein-protein interactions; binding between 2 proteins
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yeast 2 hybrid assay: The transcription factor is split into __
- DNA binding domain & RNA polymerase binding domain
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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)
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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.
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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
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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.)
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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.
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metabolome
the complete collection of metabolites present in a cell or tissue under a particular set of conditions
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metabolic flux
- the rate of flow of metabolites through the network of pathways that make up the cellular biochemistry
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metabolic engineering
Changes are made to the genome by mutation or recombinant DNA techniques in order to influence the cellular biochemistry in a predetermined way.
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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.)
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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
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EMSA: __ before mixing it with DNA increases the size of the complex.
- Adding an antibody to the protein
- → slower movement → supershift
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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.
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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.
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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
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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
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ChIP (chromatin immunoprecipitation) assay can identify __
the fragment of the DNA that is occupied by the protein within the cell
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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.
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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
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