Project weeks 2 and 3 presentation

1. UPDATE (8TH – 21ST)
JAKE HUNTER

2. ELISA PRINCIPLE
• Indirect ELISA – stripwells binding positively charged
biomolecules.
• Tetramethylbenzidine (hydrogen donor) oxidized by Horse
Radish Peroxidase. Product - Tetramethylbenzidine diimine
(detected at 450nm)

3. ELISA CONCENTRATION CURVE

4. ELISA OPTIMISATION – CARBONATE BUFFER
• Concentration range – 0.025μg/mL – 10.00μg/mL
• Graph determined optimum – 0.68μg/mL

5. ALTERED INCUBATION TIMES – CARBONATE
BUFFER

6. ELISA OPTIMISATION – CARBONATE BUFFER
• Concentration range – 0.10μg/mL – 5.00μg/mL
• Graph determined optimum – 1.23μg/mL

7. ELISA OPTIMISATION – CARBONATE BUFFER
• Concentration range – 0.025μg/mL – 10.00μg/mL
• Graph determined optimum – 0.38μg/mL

8. ELISA OPTIMISATION – HUMAN SERUM
• Concentration range – 0.10μg/mL – 5.00μg/mL
• Graph determined optimum – 1.23μg/mL

9. ELISA OPTIMISATION – HUMAN SERUM
• Concentration range: 0.5μg/mL - 15μg/mL.

10. ELISA OPTIMISATION – HUMAN SERUM
• Concentration range - 0.0128μg/mL - 250μg/mL.

11. ELISA OPTIMISATION – HUMAN SERUM
(VENOM 1)
• Concentration range - 0.1μg/mL – 10.0μg/mL

12. ELISA OPTIMISATION – HUMAN SERUM
(VENOM 2)
• Concentration range - 0.1μg/mL – 10.0μg/mL

13. SDS-PAGE PRINCIPLE
• Proteins denatured to primary structure by boiling + SDS.
• SDS provides proteins with negative
charge – attracted to anode.
• Low molecular weight pass through
Pores more easily.

14. SDS-PAGE BSA STANDARDS

15. SDS-PAGE VENOM SAMPLES

16. SDS-PAGE VENOM SAMPLES

17. SDS-PAGE VENOM EXTRACT

18. PLAN
• Begin optimising primary antibody and secondary antibodies in
ELISA.
• Run extract through SDS-PAGE and Western blot.
• Use Western blot to detect antibody binding site with different
concentrations.