L3 - More Biochem Techniques Flashcards
how do you monitor protein overexpression?
couple DNA for protein of interest to DNA for a tag protein
- transfect the DNA into cells
- fusion protein produced by cells
- can be visualized by fluorescence microscopy
- tagged proteins can also be recovered w biochem techniques
cell lysis
extract all proteins in cell using a buffer
SDS-PAGE
separate proteins according to MW
what kind of proteins are separated in SDS-PAGE?
denatured proteins
how are proteins denatured in SDS-PAGE?
1) SDS detergent
2) heating
what kind of gel used in SDS?
polyacrylamide gel
when do you use loose gels in SDS-PAGE?
allow heavy proteins through (don’t discriminate light proteins well)
when do you use tight gels in SDS-PAGE?
allow light proteins through (don’t discriminate heavy proteins well)
immunoblotting
separated proteins are transferred to a membrane and incubated for an Ab with protein of interest, and then conjugated with a secondary Ab that gives a signal
what kind of signal is seen in WB?
chemiluminescence
what kind of gels are used in immunoblotting?
1) PVDF
2) nitrocellulose
what is important to use in all immunoblotting experiments/WB?
protein ladder (molecular weight markers
what are the applications of WB?
1) check expression of protein
2) compare proteins of interest between cell types
3) assess cell signalling pathways under different conditions
loading control
confirm that equal amount of total protein was loaded onto each lane
when do you use loading controls?
when comparing different cell types, conditions, etc.
what are common proteins used in loading control?
1) GADPH
2) actin
3) tubulin
(basically most housekeeping proteins)
how is protein’s activity usually controlled?
its phosphorylation status
how can a protein’s phosphorylation status be assessed?
1) WB
2) phospho-specific Abs
ELISA
enzyme-linked immunosorbent assay
how is ELISA used?
detect small, soluble molecules
describe ELISA process
1) ELISA sandwich
2) measure absorbance
why is ELISA so good?
1) sensitive
2) quantitative
3) precise
4) high-throughput
how is immunoprecipitation used?
identify protein X’s interacting partners in cells
describe immunoprecipitation process
1) cell lysis
2) incubate cell lysate with bead-bound Ab against protein X
3) spin down beads (and protein X and its partners)
4) extract proteins from beads (heat)
5) separate extracted proteins via SDS-PAGE
6) blot associated proteins with Abs against suspected proteins
what is the limitation of immunoprecipitation?
does not indicate whether interactions are direct
for cells with fusion proteins, what kind of Ab can you use in IP?
Ab against its tag or protein itself
