Cytokine Practical Flashcards
What are cytokines?
Soluble proteins released by immune cells that regulate both innate and adaptive immune responses
What are two important pro-inflammatory cytokines?
IL-1 and TNF
How and where does NFkB exist?
It exists in a latent form in the cytoplasm of unstimulated cells
What is NFkB comprised of?
A transcriptionally active dimer bound to an inhibitor protein IkBa.
What happens when cells are stimulated?
IkBa is rapidly phosphorylated on two serine residues (S32 and S36) which targets the inhibitor protein for ubiquitination and subsequent degradation by the 26 S proteasome
What does the NFkB dimer do upon release?
Translocates to the nucleus activating target genes by binding with high affinity kB elements in their promoters
What is a good way to measure NFkB activtion?
By measuring the disappearance of IkBa from cells
What is the induction of IL-8 gene largely dependent on?
Transcription factors but largely NFkB dependent
IL-8 or CXCL8 is a typical chemokine in that it….?
Is released from tissues in the early phases of infection and causes chemotaxis in nearby responsive cells (neutrophils and naive T-cells)
What types of samples are used for the western blot?
Cell lysates
What type of samples are used for ELISA?
Cell supernatants
What does the western blot allow?
The detection of a protein of interest using a specific antibody to that protein
What is SDS page used for?
To fractionate the protein from a compex mixture
What does the reducing agent (dithiothreitol) do?
Reduces protein disulphide bridges
What does SDS do?
Coats the protein with negative charge so that the protein migrates through an electric field at a rate proportional to its size
