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Summary book Principles and Techniques of Biochemistry and Molecular Biology
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By: carolienversteijnen • 2 year ago
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IntroductionExam: book
Lab journal vs lab report
• Lab report : formal summary of activities & findings of experiment (grading)
Grade
• 70% lab report
• 30% exam
Lab journal (written)
,Lab report (typed)
- Dont search additional literature (e.g. google scholar)
Module 1: Western Blotting (HAP)
Western blotting 2 types of muscle tissue to determine myosin protein content in each of the tissue
samples.
General principles
,Electrophoresis => mitigation of charged particles under influence of electric field
• Particles move to cathode (+) or anode (-) -> depends on nature of their net
charge
- Many important molecules possess ionisable groups & are electrically
charged
- Amino acids, peptides, proteins, nucleotides & nucleic acids
• Equipment:
- Power pack
- Electrophoresis unit
• Electrophoresis unit for running 2 types of gel systems:
- Vertical slab gel => gel formed between 2 glass plates (separate
proteins in acrylamide gels)
- Horizontal gel => gel cast on glass or plastic sheet & placed on
cooling plate
• Buffer => maintain constant rate of ionisation of molecules being
separates
Equations: how do charged species separate?
• Potential difference (∆V) => generates potential gradient (Ē)
CL
Western blot (WB) => tool to separate & analyse proteins
• Separate proteins based on size
• Main principles
- Detection & separation of protein based on molecular weight
- Identify presence of specific protein & determine the level of relative expression in
sample
- Semi-quantitative technique => provides relative comparison of protein levels, no
absolute measure of quantity
, Work flow
1. Preparation of samples
2. Protein extraction
3. SDS-PAGE
4. Protein transfer
5. Blocking & antibodies incubation
6. Detection
Preparation of samples (1)
• Protein from 2 different murine muscles from mice
- Extensor digitorum longus (EDL) & soleus
• Check expression of myosin
Protein extraction (2)
Proteins collected in lysis buffer containing
proteinase inhibitors
SDS-PAGE (3)
• Dodecyl sulphate
polyacrylamide gel
electrophoresis
• Electrophoresis =>
describes mitigation of charged particle under influence of electrical
field
- 2-mercaptoethanol => break H2S bridges to unfold proteins
- SDS has – charge -> make whole protein negative
- Proteins will move to + pole
- Proteins with high molecular weight (larger proteins) will travel
less far/fast
Protein transfer (4)
• Protein transfer = blotting
- Wet transfer or semi-dry transfer
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