Methods- Exam 2
SDS Page: To separate proteins by size. After Immunoprecipitation, SDS page separates the
DNA from the protein, denatures them, and negatively charges the protein so that it will migrate
down the poly acrylamide gel via electrophoresis, and protein can be analyzed that way. Ex.)
Taking out antigen antibody complex via Immunoprecipitation then use SDS page to separate
out desired proteins to identify it. How far it migrates can be used to compare against known
Recombinant Libraries: Collection of clones that make up a complete genome. Can use it to
isolate target genes by use of a labelled gene probe.
DNA sequencing: To identify sequences in genes. Fluorescent dye incorporated into DNA
molecules with synthesis-stopping dideoxinucleotides, fragments are separated by
electrophoresis gel, and laser excited fluorescent dye, color is analyzed to determine DNA size
therefore order DNA sequence.
DNA Microarray injection: To measure intensity, compare how much of each gene’s DNA is in
a cell to measure expression. Use mRNA from two proteins in two different plates, take copy
and use cDNA to probe different gene plates, measure expression and compare the genes
between the proteins via fluorescence.
CHECK FOR PROTEIN-PROTEIN INTERACTIONS:
• Co-immunoprecipitation is considered to be the gold standard assay for protein–protein
interactions, especially when it is performed with endogenous (not overexpressed and
not tagged) proteins. The protein of interest is isolated with a specific antibody. Interaction
partners which stick to this protein are subsequently identified by Western blotting.
[1Interactions detected by this approach are considered to be real. However, this method can
only verify interactions between suspected interaction partners. Thus, it is not a screening
approach.Anote of caution also is that immunoprecipitation experiments reveal direct and
indirect interactions. Thus, positive results may indicate that two proteins interact directly or
may interact via one or more bridging molecules. This could include bridging proteins, nucleic
acids (DNAor RNA), or other molecules.
Northern Blot: mRNA (run through gel, filter, target with labelled probe/antibody, check for
detection of RNA).
Southern Blot: DNA (run through gel, filter, target with labelled probe/antibody, check for
detection of DNA).
Western Blot: AKA immune-blotting for proteins, using SDS page, let migrate, transfer to filter,
apply a labelled antibody to filter used to detect proteins in cell extracts.
Immunoprecipitation: gel electrophoresis/gel fractionation via specific binding of
antibody/antigen complex. Checking for protein of interest in a mix of proteins. Isolation
Co- Immunoprecipitation: check what proteins are associated with it after isolation via protein
-protein interactions. Co-IP works by selecting an antibody that targets a known protein that is believed
to be a member of a larger complex of proteins. By targeting this known member with an antibody it may
become possible to pull the entire protein complex out of solution and thereby identify unknown members
of the complex. This works when the proteins involved in the complex bind to each other tightly, making
it possible to pull multiple members of the complex out of solution by latching onto one member with an
Chromatin Immunoprecipitation: Specifically for Transcription Factors. Cross link DNA and
proteins, incubate with antibody against transcription factor of interest, collect complex, separate
DNA and TF, and purify DNA to get fragments with binding sites for that TF. Then analyze DNA
via PCR/ genome wide analysis.
FRET: Check Protein-Protein Interactions. Put in 2 different fluorescent dyes in two
different proteins and when shine a specific frequency that excites through them. If shine
frequency on one of the cells, and get second dyed cell emission, then interaction has
occurred. The dye of the first protein excites the second dye if interaction has occurred,
leading to two different wavelengths.
FRAP: Check Protein Movement. Based on rate of movement, can infer where a
protein moves in the cell. If not sure location of function of protein, can dye a portion of
the protein, laser a portion of the cell, depending on how quickly the light comes back
tells how quickly the protein moves in the cell.
Pulse-Chase: a method for examining a cellular process occurring over time by successively
exposing the cells to a labeled compound (pulse) and then to the same compound in an unlabeled
form (chase). Pulse with radioactive molecule and chase with a normal molecule until pulse is
gone. Radioactively synthesize a molecule to see where it ends up. A selected cell or a group of
cells is first exposed to a labeled compound (the pulse) that is to be incorporated into a molecule
or system that is studied (also see pulse labeling). The compound then goes through
the metabolic pathways and is used in the synthesis of the product studied. For example, a
radioactively labeled form of leucine ( H-leucine) can be supplied to a group of pancreatic B cells,
which then uses this amino acid ininsulin synthesis.
Shortly after introduction of the labeled compound (usually about 5 minutes, but the actual time
needed is dependent on the object studied), excess of the same, but unlabeled, substance (the
chase) is introduced into the environment. Following the previous example, the production
of insulin would continue, but it would no longer contain the radioactive leucine introduced in the
pulse phase and would not be visible using radioactive detection methods. However, the
movement of the labeled insulin produced during the pulse period could still be tracked within the
cell. Mass spectrometry: Identify unknown compound. Breaks up substituents and
view remaining parts of the molecule, then can compare remaining parts of
molecule and their masses to determine the molecule. Analyze parts of molecules
based on isotope abundances. Commonly used for proteins
Sub-Cellular Fractionation: Separating subcellular structures by size or density.
Gel Fractionation: Isolating proteins of interest via pulling down a complex if SDS.
Breaking apart nucleic acids if agarose.
Velocity Centrifugation: sucrose gradients to separating molecules by rates of
PCR: Using specific known primers to amplify a gene of interest. Primers on either side
of gene then TAC polymerase elongates sequence and repeats to amplify one specific
Plasmid Vectors: Clone a gene and insert it into plasmid vector for gene expression.
Homologous Recombination: Adding a similar but inactive sequence that binds to
regular DNA and expressing it to see if that gene you replaced is involved with a certain
type of expression.
Electro mobility Shift Assay: for TF binding sites. Radio labelled DNA half incubated with
sequence specific protein, analyze by electrophoresis and non - denaturing gel, protein
DNA complex intact, protein binding detected by slower migration, therefore can
compare sequence with free DNA.
DNAAffinity Chromatography: To isolate known protein (If known DNA and protein that it
goes with) Purifying proteins from a mixture of proteins. Add oligonucleotides with
specific sequence to agarose beads, put into column, add mixture of cell proteins,
protein of interest will bind to DNA, and will stay in column and then separate DNA from
proteins to purify.
DNase I split printing: Helps determine specific sequence on a fragment where a known
protein binds. Makes footprint on gel after fragment has been cut, can compare two
samples with (cut and uncut, not protein and with protein) to determine sequence on a
fragment. Fragments bind protein to half, add endonuclease that makes cuts, denatures
and run through gel, see DNA where cuts are not made due to binding of proteins.
In-vitro Splicing: To use exons for other experiments. Cloned intron in plasmid is
transcribed to produce pre-mRNA. Nuclear extract splices produce exons in vitro.
Radioactivity: Measures amount of newly synthesized proteins or nucleic acids in
UV absorption: determines amount of already present nucleic acids in extract.
Post Transcriptional Gene Silencing: double stranded RNA interference w/ sense and
antisense RNA In Situ Hybridization: hybridization in intact cells.
Genome –Wide Analysis: After chromatin Immunoprecipitation, can use PCR (individual
binding sites), microarrays (use with chromatin Immunoprecipitation) and lasers through
computer to check a whole sequence.
HETEROCHROMATIN VS EUCHROMATIN : Heterochromatin appears as small, darkly staining,
irregular particles scattered throughout the nucleus or accumulated adjacent to the nuclear
envelope. Euchromatin is dispersed and not readily stainable. Euchromatin is prevalent in cells that
are active in the transcription of many of their genes while heterochromatin is most abundant in
cells that are less active or not active.
Map Kinase (RAS/RAF/MEK/ERK):
1.) Growth factors causes dimerization of 2RTK’s auto phosphorylate. Auto
phosphorylation allows SH2 domain containing proteins to bind. Once SH2
domain proteins bind, the GEF binds to the SH2 domain contain