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Effects of Traditional Chinese Medicine on Toll-Like Receptor 4 of Carsinoscorpius rotundicauda

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). Introduction: Toll-like Receptors. Transmembrane proteins

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Effects of Traditional Chinese Medicine on Toll-Like Receptor 4 of Carsinoscorpius rotundicauda

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  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. 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.

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

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

  5. Procedure—Running SDS-PAGE • 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

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

  7. 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

  8. 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

  9. 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

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