Chapter 15 Quiz Flashcards
JAK-STAT pathway
cytosines and growth factors bind to JAKs/Janus Kinases (transmembrane receptors)
When cytosine binds to its receptor, JAKs transfer a phosphate group to other proteins. The phosphate group causes a conformational change in STATs. When STAT proteins bind to a receptor, JAKs add another phosphate group to the STAT proteins, causing them to dissociate from the receptor and bind to each other. STAT dimers translocate to the nucleus and activate target genes.
Antigens
Antigens: foreign proteins
Antibodies
proteins used to detect antigens in immune system
recognizes certain region of antigen known as “epitope”
Monoclonal or Polyclonal
- monoclonal: identical, recognizes same epitope
- polyclonal: mixture of several antibodies, recognizes different epitopes of the same antigen
Aptamers
alternative to antibodies
They are nucleic acid or peptide-based molecules that bind to a specific target molecule
Purifying proteins and two common methods?
Purifying proteins: allows generating antibodies, determining protein’s sequence and determine a protein’s binding partners
Two common methods:
- Chromatography
- Immunoprecipitation (IP)
Chromatography
Allows mixture of proteins in a solution to be separated on a column
Gel-filtration Chromatography
proteins are separated based on their size
Ion-exchange Chromatography
proteins are separated based on their charge
Affinity Chromatography
uses small molecules, fixed to the matrix, to bind to proteins that may have high affinity
Immunopreceipitation (IP)
uses antibodies to purify protein of interest out of solution
Western Blot (WB/Immunoblot)
Most common used method to measure expression of a protein in a tissue of cell sample
SDS-PAGE
Sodium dodecylsulfate - polyacylamide gel electrophoresis
It’s a negatively charged detergent that binds to hydrophobic regions of the protein, allowing it to become soluble
How do smaller and larger proteins travel?
Larger proteins travel slowly
Smaller proteins travel quickly
Cell fractionation
Used to see where in cells the protein is localized
Enzyme-linked immunoabsorbent assay (ELISA)
used to measure expression of a protein
Alternative to a WB
Radioimmunoassay (RIA)
measures very low concentrations of proteins
Immunohistochemistry (IHC)
used to determine the spatial expression pattern of a protein
Immunoelectron microscopy (IEM)
Used to visualize protein expression within subcellular structures
- can show protein is localized in a particular subcellular compartment
Reporter Protein
attached to proteins of interest and allows determining expression/location of a protein in living cells
Co-immunoprecipitation
determines if a protein physically interacts with another protein
Protein Affinity Chromatography
used to isolate and identify proteins that physically interact with each other
Mass Spectrometry
determines the identities of potential binding partners
- identify and sequence proteins by breaking them apart and determining the mass to charge ratio of their fragment
Mass spectrometry
determines identity of binding partners
Yeast two-hybrid assay
investigates protein-protein interactions
In normal cell: GAL4 binds to UAS
Gal4: transcription factor
UAS: promoter region (upstream activating sequence)
Posttranslational modification (PTM)
modification that happens to one or more aminoacids on a protein after it’s translated by a ribosome
Kinase Assay
determine whether one protein is capable of phosphorylating another protein
Methyltransferase or Acetyltransferase Assays
determines if protein is capable of adding a methyl or cetyl group to a target protein
Electrophoretic Mobility Shift Assay
used to determine if a protein is directly able to interact with a short specific sequence of DNA
Chromatin Immunoprecipitation (chIP)
shows if protein interacts with specific region of DNA
chIP on chip assay
Determines if all DNA sequences are bound to protein
DNA sequences from chIP expt are run on a DNA microarray
Luciferase Assay
determines if protein can activate or repress expression of target gene
establishes functional connection between protein and amount of gene product made
Luciferase
enzyme used for bioluminase
If protein upregulates transcription of target gene: cells express luciferase
If protein downregulates transcription: expresses less luciferase than normal
Next generation sequencing (NGS)
makes sequencing large stretches of DNA cheaper and faster
Hybridization
phenomenon whereby two complementary nucleic acid strands bind to each other
Southern Blot
identifies if known DNA sequence is present in a sample by using a specific prob to identify the sequence
Northern Blot
identifies presence of RNA
