Effects of Traditional Chinese Medicine on Toll-Like Receptor 4 of Carsinoscorpius rotundicauda
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Transcript of Effects of Traditional Chinese Medicine on Toll-Like Receptor 4 of Carsinoscorpius rotundicauda
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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 Analysis
Introduce 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 Results
ladder 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