Molecular methods in SKM Flashcards
Explain the steps of protein extraction
- Tissue homogenistaion - polytron/scissors / sonication
- Cell fractionartion - differential centrifugation - validation
- Phosphatase inhibitors - do you need them, are they appropriate for your targets
- Buffers (EDTA, EGTA (Ca2+), detergent (TX-100)
- Protein quantification assays - interfering blood/chemistry
- Load the protein, mix with sample buffer
- Tris-HCL, glycerol (Density), bromophenol blue (tracking dye)
- Reducing agents (DTT) in sample buffer breaks bonds - boiling/heating
- SDS disrupts hydrophobic areas and coat proteins with (-) charges which overwhelms charged R groups (+). WILL MOVE FROM ANODE TO CATHODE
What are the basic principles of westernblotting?
-Detects a single protein from ~30,000 in a complex mixture
- protein extraction to isolate cellular proteins
- Quantify protein- load equal volume
- Transfer to membrane blocking - reduce non-specificity (gel electrophoresis)
- Primary antibody - Detect specific epitope bind to 1(primary)
- 2ary conjugated antibody washes- reduce non-specificity
- Exposure to ECL- chemiluminescence
- - Quantification -
Describe SDS-Polyacrylamide Gel Electrophoresis
- Resolve proteins by mass
- Load samples in gell wells
- Mercaptoethanol denatures protein
- SDS binds protein stochiometrically
- Small molecule moves more quickly through gel, Large molecule moves more slowly through gel
What is the purpose of gel transfer in blocking and electro-blotting?
- The gel it is being transferred to has a greaterr affinity for antibodies
What are Monoclonal antibodies?
- produced by one type of immune cell and are all clones of a single parent cell
What are polyclonal antibodies?
A mixture of immunoglobulin molecules secreted against a specific antigen
What are phsopho-specific antibodies?
- Recognise epitopes only when phosphorylated at specific residues
What is “stripping” the membrane?
- Removal of antibodies from the membrane using either pH, heat or detergent
- Glycine, 2-mercaptoethanol or commercial reagents used
- These may strip the antigen
- Stripping success is checked by exposing to ECL reagents and secondary antibodies following stripping to validate both antibodies removed
What is immunoprecipitation?
- Add antibody against protein of interest
- Antibody binds to protein of interest
- Adding protein A or G makes antibody-protein complexes insoluble
- Centrifugation of solution pellets the complexes, removal of supernatant and washing
How can you calculate fold increase from western blotting results?
Corrected fold increase is half the expeccted one
Ratio target gene in experimenntal/comtrol = fold change in target gene/ fold change in ref gene
What is the goal of measuring gene expression?
To measure effects of an intervention on the expression of a gene, i.e.m its transcription
What are other methods for measuring mRNA abundance?
- Traditional thermal cycling
- Northern blotting
- Microarray technology
- Real time PCR is an advanced method of measuring mRNA abundance - excellent sensitiviy, versatility and simplicity
What is reverse transcription?
- Input RNA (TOTAL)or poly A isolated mRNA
- Oligo
- Random hexamers
- Gene specific
- Retroviral reverse transcriptase
(RT) enzyme (e.g. MMLV)
Adds a bias to results
Efficiency usually not known
What is a Polymerase Chain Reaction?
- Polymerase enzyme
- Amplifies DNA
- Fluorescent dye such as SYBR green (binds to dsDNA)
- Deoxyribonucleotides (dNTPs)
- ## Gene specific primers (derived bio-informatically)
What are the conditions for thermal cycling?
2 phases
- annealing/elongation and melting
- Each stage represents 1 cycle
- 40 cycles per run
