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proteomics method that separates proteins by their charges (isoelectric focusing) & then separating them by weight
2D gel electrophoresis
Explain the isoelectric focusing step during 2D gel electrophoresis
The gel shows a gradient of pH. Each protein on the gel will migrate to its isoelectric point (a specific pH for each point), which is the point where it loses its charges and becomes neutral.
During 2D gel electrophoresis, the gel containing the protein bands is cut and soaked in __ and rotated by __ degrees
sodium dodecyl sulfate+2ME; 90
steps in 2D gel electrophoresis
- 1st dimension: Proteins are loaded & separated by their isoelectric points.
- The strip of gel containing the protein bands is cut & soaked and rotated by 90 degrees.
- 2nd dimension: A second electrophoresis (in a denaturing gel) separates the proteins by their molecular weights (with SDS & BME)
problems with 2D gel electrophoresis
- Protein samples may have a presence of hydrophobic & non-soluble proteins (This means some will have no charge.)
- low reproducibility
- difficulties in analyzing & comparing 2 proteomes
To compare 2 samples of proteomes from 2D gel electrophoresis, it is possible to __
- label the constituents of 2 proteomes with different fluorescent colors (ex. one sample all proteins green, the other sample all proteins red) & then run them together in a single 2D gel.
- Visualization of the 2D gel under different lights enable the intensities of equivalent spots to be compared (2 different colors for each spot, red from one sample & green from the other sample)
After 2D gel electrophoresis, a spot of protein is excised from the gel and __
digested with trypsin to produce a series of peptides before ionization (through MALDI-TOF)
During MALDI-TOF in the mass spectrometer, __
(Matrix-assisted laser desorption ionization time-of-flight)
The peptides are ionized by a pulse of energy from a laser and then accelerated down the column to the reflector onto the detector. Data is visualized as a spectrum.
The time-of-flight of each peptide depends on its __
mass to charge ratio
By using a database of __, it is possible to determine the identity of each pick of the MALDI-TOF spectrum.
predicted molecular masses
To determine the function of a protein, it is important to __
identify what other proteins it interacts with & the consequences of that interaction. DNA cloning & cDNA libraries can help to determine the function.
step of DNA cloning
- 1. recombining each DNA fragment into a cloning vector (plasmid)
- 2. propagating each vector-insert (DNA fragment) in an organism (most of the time E. coli) - Each colony of bacteria will contain the cloned DNA fragment
Define a DNA library
There is a collection of different cloned DNA fragments in one colony of bacteria. A library contains many colonies of bacteria.
In a DNA library the __ is the same in all, but the __ mostly is unique for each colony.
vector (plasmid); insert (DNA fragment)
How is a cDNA made?
- Most eukaryotic mRNAs have a poly(A) tail at their 3' end.
- With RNA as the template, a poly-T primer & reverse transcriptase can make a complementary strand to the mRNA, known as the cDNA.
A cDNA represents what genes were expressed in the cell.
Once a cDNA is made, how is a cDNA library made?
- The RNA component of RNA-DNA is degraded.
- The second strand of cDNA is made. Each one of these cDNA can be cloned (inserted into a vector & propagated in bacteria). A cDNA library is made.
A cDNA library is a collection of all these cDNA clones generated from a particular isolated total mRNA from the cell.
producing a phage display library step 1
cDNA is cloned and fused with the capsid & inserted into bacteria. A cDNA library (representing different coded proteins in for example a human cell) is made into phage vectors in fusion with the viruses' coat gene and then infecting bacteria with these constructs. Each phage particle produced by these transformed bacteria therefore display one of these human proteins on their coats.
producing a phage display library step 2
The protein of interest is immobilized within wells of a microtiter tray and the phage display library (many phages, each one has one type of human protein on its surface) is added to the wells. After washing, the phages that are retained in the well are those displaying a protein that interacts with the protein of interest.
producing a phage display library step 3
The retained phages can be isolated and their chimeric "coat gene DNA - human cDNAs" can be sequenced to identify what protein was interacting with the protein of interest.
After sequencing, the cDNA (from recombinant DNA) can be used to identify the protein that interacts with the protein of interest.
steps in Yeast Two Hybrid Assay
- The DNA binding domain interacts with the RNA polymerase binding domain. Activation (transcription) occurs and the blue gene is expressed.
- They tested out interacting proteins by separating the 2 domains. They tried binding the protein of interest to the DNA binding domain and then another protein with the RNA polymerase binding domain. When putting the 2 together, the transcription of the blue gene becomes activated.
two-hybrid protein interaction image
Changes are made to the genome by mutation or recombinant DNA techniques in order to influence the cellular biochemistry in a predetermined way
Electrophoretic Mobility Shift Assay (EMSA) can be used to show __
interaction of a particular protein with a DNA fragment
- DNA is radioactively labeled & placed in 2 tubes. In one of the tubes, a protein is added. The DNA contains a binding site for protein.
- Acrylamide gel electrophoresis & autoradiography is used to visualize bands.
- DNA will show as a single band (corresponds to the size of the DNA fragment) and DNA with a DNA binding protein will show 2 bands. Adding an antibody to the protein before mixing it with DNA increases the size of the complex, creating slower movement. This is called a supershift.
How is a supershift in EMSA generated?
by mixing the target DNA, the protein, & an antibody specific to the protein
nuclease protection foot printing steps
- DNA is in one tube and DNA + protein is in the other tube. DNAse is added to both tubes Protein binding to DNA protects the DNA from digestion.
- On the DNA-only side, DNA molecules are cut at random by DNAse & separated by size using gel electrophoresis. On the protein-bound side, DNA molecules are first bound to a repressor & then subjected to DNAse treatmeat.
- The footprint indicates where proteins were bound.
Nuclease protection foot printing image
The stars indicate the radioactive labels at one side of the fragment. The arrows show where DNAse cuts. The red circle indicates the protein (lac repressor) that is bound to the DNA
method that helps to identify which part of the DNA structure is necessary for protein to bind to the DNA
chemical interference foot printing
steps of chemical interference foot printing
- DNA is chemically modified either at the phosphate, sugar, or bases, and then mixed with the protein of interest.
- Foot printing assay is performed. (See nuclease protection foot printing).
- The foot print will disappear in the gel shift if the protein no longer binds to the DNA after modification.
The part of the DNA that is necessary for protein binding is determined by __
A foot print disappearing in areas where there was a protein bound (was a footprint). This means the chemical modification of this part of DNA stopped protein from binding.
The green star indicates chemically modified DNA