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

rotundicaudaMentored 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)

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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.

3Introduction: Toll-like Receptors

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Source: http://www.cat.cc.md.us/courses/bio141/lecguide/unit1/prostruct/toll/lps.html

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Separate hemocytes and cell-free

hemolymph Lyse hemolymph

SDS-PAGE

Western Blot AnalysisIntroduce TCM and bacteria (Pf) to

Horseshoe crabs (Injection)

Extract blood

Standard Procedure

SDS-PAGE Analysis

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

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SDS-PAGE Resultsladder TCM #1 TCM #2 TCM #10 TCM #11

TCM #2 + bact

Naïve 1 Naïve 2

85kDa

60kDa

50kDa

40kDa

25kDa

20kDa

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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

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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

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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