1. 1 Effects of Traditional Chinese Medicine on Toll- Like Receptor 4 of Carsinoscorpius rotundicauda Mentored by: Ms. Sandra Tan Co-Mentored by: Mr. Goh Yan Yih Tay Jia Xi (3I/28)Tan Wei Jie Eugene(3I/26)Ang Dun Yang (3J/01)

2. 2 Introduction: Toll-like Receptors Transmembrane proteinsTransmembrane proteins Plays a major role in:Plays a major role in: –Pathogen recognition through the detection of Pathogen-associated molecular patterns (PAMPs). –Recognition of endogenous ligands Initiates inflammatory and immune responses.Initiates inflammatory and immune responses.

3. 3 Introduction: Toll-like Receptors Source: http://www.cat.cc.md.us/courses/bio141/lecguide/unit1/prostruct/toll/lps.html

4. 4 Separate hemocytes and cell-free hemolymph Lyse hemolymph SDS-PAGE Western Blot Analysis Introduce TCM and bacteria (Pf) to Horseshoe crabs (Injection) Extract blood Standard Procedure SDS-PAGE Analysis

5. Procedure—Running SDS- PAGE 10µl of each sample loaded10µl of each sample loaded Gel was ran for 50min at 20mA(constant current) until dye front reach lower edge of the gelGel was ran for 50min at 20mA(constant current) until dye front reach lower edge of the gel Stained with Coomassi blue overnightStained with Coomassi blue overnight 5

6. SDS-PAGE Results 6 ladderTCM #1TCM #2TCM #10TCM #11 TCM #2 + bact Naïve 1 Naïve 2 85k Da 60k Da 50k Da 40k Da 25k Da 20k Da

7. Procedure—SDS-PAGE Gel Analysis(MALDI-TOF) The proteins is digested by trypsin to form peptidesThe proteins is digested by trypsin to form peptides Peptides are mixed with the matrix solutionPeptides are mixed with the matrix solution –To protect the peptides from the laser Laser is used to ionise the peptides into positively charged ionsLaser is used to ionise the peptides into positively charged ions The peptides is put to fly through a vacuum tube with two metals plates, of opposite chargeThe peptides is put to fly through a vacuum tube with two metals plates, of opposite charge 7

8. Procedure—SDS-PAGE Gel Analysis(MALDI-TOF) The peptides, being positively charged, will hit the negatively charged metalThe peptides, being positively charged, will hit the negatively charged metal Hence, registering on the computer.Hence, registering on the computer. The difference in mass of the peptides will causeThe difference in mass of the peptides will cause –Heavier peptides to fly slower –Lighter peptides to fly faster 8

9. Procedure—SDS-PAGE Gel Analysis(MALDI-TOF) Readings on the computer willReadings on the computer will –Show the lighter peptides first –Show the intensity of the impact which is the number of peptides with the same mass hitting the metal plate together 9