Lab Practical-Cytokine Practical

how does NFkB work? how is it activated?

• p50/65 heterodimer

• IL-1 activated NFKB through a kinase cascade

• IkB becomes phosphorylated and translocates to the nucleus

where is IkB phosphorylated? what occurs after this?

• on 2 serine residues S32 and S36

• Targets the inhibitory protein for ubiquitiniation

what does the Western blot show?

• shows that IkK undergoes autophosphorylation by IL-1 and activates NFKB

• MEASURING THE DEGRADATION OF IKK at different timepoints

what does the ELISA measure?

• the production of IL-8 measuring if NFKB translocates to the nucleus and makes IL8

what is a sandwich ELISA? why’s it called this?

• capture antibody locks the target antigen to the plate

• detection antibody is then added for specificity of detection

• HRP is added- for colour detection. it is an enzyme that binds to the detection antibody- colour change is proportional to the amount of cytokine produced

in SDS-PAGE and western blotting- what is FCM?

fatal calf serum- gives growth factors

what is SDS-PAGE? what does DTT and SDS do?

• fractionate the protein from a complex mixture- such as cell lystate

• lysate proteins are denatures by boiling sample buffer with a reducing agent and SDS

• DTT specifically- reduces protein disulphide boridges

• SDS coats the protein with a negative charge- so it migrates through a electric field in proportion to its size?

how are proteins transferred?

• electrophoterically to nitrocellulose membranes

• membrane is soaked a blocking solution to occupy all binding sites

• membrane is then incubated with specific antibody for the protein of interest

• then secondary antibody with a reporter enzyme such as HRP or IRDeye dye

• allows visualisation of protein band

what is in the cell lysate?

benzonase- degrades DNA and RNA

what is the blocking solution? what method is this for?

• BSA- bovine serum antibody

• In a sandwich ELISA, Bovine Serum Albumin (BSA) is primarily used as a blocking agent to prevent non-specific binding of antibodies to the microtiter plate, which reduces background noise and increases assay sensitivity. It fills empty, reactive sites on the plastic surface after the capture antibody is immobilized. 

what is the secondary antibody?

• it recognises mouse IgG- goat

• stops background noise

if you’re given 50ng/ml and need to dilute this to 2000pg/ml, how would you do this in a final volume of 400micro;

5ng/ml is 50000pg/ml

50000/2000=25, 1 in 25 dilution

400microL/25= 16microL stock and 384microL diluent

if you’re given 600ng/ml and you need it to 10ng/ml and need a total volume of 2500microL of detection antibody

600/10=60

2500/60=42 microL