Future Prospects

Question 1

How could we test for his expression on the virus?

Answer 1

NanoSight
Western blotting
His tag elisa - chosen because of our antibody.

Question 2

Taqman qPCR

Answer 2

RT used to form cDNA.
Samples are loaded with probes and primers.
The probes contain a 5’ reporter and 3’quencher.
They bind during the polymerisation step.
When the PCR reaction takes place the probe is cleaved activating the reporter.

Question 3

How might the beads experiment be conducted?

Answer 3

Load the beads into a column. 
Equilibrate using PBS
Feed in aliquot of each virus
Take samples periodically. 
Test samples for virus in the flow through using taqman qPCR.

Question 4

How might we determine if the His tag is removed?

Answer 4

Process virus aliquots as if to remove free His. Trypsin. Micron YM100.
Create assay with dilution series of HT2.
Apply in triplicate to ELISA plate.
Block
Apply primary made up in blocking buffer.
ELISA or western blot and microfiltration.

Question 5

Western blot for His tagged antibody.

Answer 5

Apply virus to gel.
Sandwich gel between fibrous sheet, blotting paper, gel, nitrocellulose paper, blotting paper, fibrous sheet.
Run on electrophoresis.
Isolate nitrocellulose and apply 3 step Ig stain. Using enzyme this time.