in vitro experimentation; proteins, DNA-proteins interactions Flashcards
how can we separate proteins?
using SDS-PAGE
how can we detect proteins?
using western blot, immunofluorescence, immunohistochemistry, ELISA and FACS
how can we study protein complex interactions?
by using co-immunoprecipitation
how can we study DNA-protein interactions?
by using luciferase assats, EMSA and ChIP
priniciple of SDS-PAGE
separation of proteins based on the molecular weight
principle of Western Blot, ELISA, IHC, IF and FACS
detection of proteins using specific antibodies
what is SDS-PAGE?
Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis
general process of SDS-PAGE
- proteins are negatively charged (due to SDS) and move to positive electrode
- proteins separate by size (molecular weight)
- smaller proteins move faster
what is needed to carry out an SDS-PAGE?
- acrylamide gel
- protein sample
- loading dye (density + dye)
- SDS-based solution
how can we identify the proteins when we remove the gel from the tank?
using a dye of Coomassie Blue Stained
using antibodies to detect proteins
- antibodies vary in specificity but ideally they only recognise one epitope
applications for antibodies
basic research, diagnostics, therapeutics
process of western blotting
- isolate all proteins form sample
- gel electrophoresis
- transfer to membrane
- block
- primary antibody
- secondary antibody
three ways of protein detection in western blot
- colorimetric
- chemiluminescence
- fluorescence
what is the ELISA assay?
enzyme-linked immunabsorbent assay
what are the 4 types of the ELISA assay?
- direct ELISA
- indirect ELISA
- sandwhich ELISA
- competitive ELISA
what are FACS?
fluorescence-activated cell sorting
prinicples of FACS
(single cell analysis)
- specialised type of flow cytometry
- provides method for sorting heterogenous mixture of biological cells into two or more containeers, one cell at atime, based upon the specific light scattering and fluorescent characteristics of each cell
- allows single cell separation and recovery
what is co-immunoprecipitation?
a protein complex that can be isolated from a protein mixture by using an antibody that is specific for one protein of the complex
what is luciferase reporter assay used for?
for transcription factor activity
what is EMSA used for?
electrophoretic mobility shift assay
- determines if a protein or mixture of proteins is capable of binding to a given DNA or RNA sequence
what is ChIP-Seq used for?
combines chromatin immunoprecipitation (ChIP) with a massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins
luciferase reporter vector
sequence of luciferase protein= emits light
- introduction of promoter, if active = transcrip/trans of luciferase protein = light
light measured in luminometer
reaction components of EMSA
- protein
- specific competitor
- mutant/non competitor
- probe
- antibody
process of ChIP-seq
- TFs bound to DNA in nucleus
- fix the protein to the DNA
- immunoprecipitate the TF of interest, along with the attached DNA
- sequence the DNA to determine the binding site
summary of protein analysis techniques
- antibodies very useful in lab for identification of proteins in WB/ELISA (cell lysates), immunofluorescence/FACS (cells in culture) and immunohistochemistry (tissues)
- protein complexes can be isolated from a protein mix. by using antibody speciic for one protein of the complex (co-immunoprecipitation)
- DNA-protein interactions are critical for gene expression regulation & we can study them with techniques such as luciferase assay, EMSA, and ChIP-Seq
