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Effects of Traditional Chinese Medicine on TollLike 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) 1 Ang Dun Yang Introduction: Toll-like Receptors • Transmembrane proteins • 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. 2 Introduction: Toll-like Receptors Source: http://www.cat.cc.md.us/courses/bio141/lecguide/unit1/prostruct/toll/lps.html 3 Standard Procedure Introduce TCM and bacteria (Pf) to Horseshoe crabs (Injection) SDS-PAGE Analysis Western Blot Analysis Extract blood SDS-PAGE Separate hemocytes and cell-free hemolymph Lyse hemolymph 4 Procedure—Running SDSPAGE • 10µl of each sample loaded • Gel was ran for 50min at 20mA(constant current) until dye front reach lower edge of the gel • Stained with Coomassi blue overnight 5 SDS-PAGE Results ladder TCM #1 TCM #2 TCM #10 TCM #11 TCM #2 Naïve 1 + bact Naïve 2 85k Da 60k Da 50k Da 40k Da 25k Da 20k Da 6 Procedure—SDS-PAGE Gel Analysis(MALDI-TOF) • The proteins is digested by trypsin to form peptides • Peptides are mixed with the matrix solution – To protect the peptides from the laser • Laser 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 charge 7 Procedure—SDS-PAGE Gel Analysis(MALDI-TOF) • The peptides, being positively charged, will hit the negatively charged metal • Hence, registering on the computer. • The difference in mass of the peptides will cause – Heavier peptides to fly slower – Lighter peptides to fly faster 8 Procedure—SDS-PAGE Gel Analysis(MALDI-TOF) • Readings 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