Trigger 8 Flashcards
what is IGF
a signalling molecule that binds to tyrosin kinase molecule
desrcibe the IGF/Akt pathway
Growth factor binds to tyrosin kinase molecule
receptor dimersiation and conformational change
transautophossphorylation
sticky patches attracts SH2 of PI3K
phosphorylates PIP2 to PIP3
PIP3 recruits protiens with particular binding sites - PH
PIP3 recruits PDK1
phosphorylates Akt
Akt binds to PIP3
Akt further phosphorylates mTOR
Akt now super active, phosphorylaets others
kinase cascade
what does mTOR stimulate
mTOR2 rictor –> activates Akt
mTOR1 raport –> S6 kinase –> S6 ribosome protein
Akt indirectly activates mTOR1
phosphorlaytes and inactiavtes Rheb-GAP
inhibited Tcs2 prevents Rheb-GTPase activity
activated Rhed-GTp bound activates mTORC1
what does Akt inhibit
inhibits FOXO - transcription factor
- genes involved in autopahgy and protein degredation
inactives Bad
- bad is part of a complex with a protein which inhibits apoptosis, by inhibiting Bad, it allows the protein to be active
advantages on one step RT-PCR
simplifies work flow
reduces variation
minimises possible contianination
process a larger number of samples
disadvantages of one step RT-PCR
uses gene specific primers for amplification, limiting to a few genes per sample
advantagef of two step RT-PCR
usefult for detecting multiple genes
seperation of reaction allows for optimisation of reaction conditions for each sample
flexibilty with reverse transcriptase priming and PCR set up
disadvantages of two step RT-PCR
includes mulitple steps for en extended work flow
has additional sample hloding and processing, increasing the chance of contamination and variation in results
what is ELISA
enzyme linked immunosorbet assay
plate based assay technique
designeed for directing and quantifying substances such as peptides, proteins and antibodies
how does ELISA work
performed in 96 well polystyrene plates which passovely bind antiobodies and proteins
binds and immobilises reagents
easy to seperate bound from non bound
detection enzymes can be linked directly to antibody or introduced through a secondary antidofy that recognises the primary antibody
what is an indirect assay
uses a labelled secondary antibody for detection
has specificty for the primary antibody
advantages of indirect assay
wide varity of labelled secondry antibodies are available
verstile
increased sensitivity
disadvantages of indirect assay
cross reactivity might occur with the secondary antibody
extra incubation step is required
what is a direct assay
labelled primary antibody that reacts directly with antigen
advantages of direct assay
quick beacuse only one antibody and fewer steps
no cross reactivity
