Download PAGE n MALDI for reference - AOS-HCI-2011-Research

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