Specific Laboratory Techniques Flashcards
What sample type does immunohistochemistry chemistry require?
- Very thin section of tissue (frozen or embedded in paraffin).
- Antigens visible on tissue slice
Describe direct immunohistochemistry
- Buy antibody specific for protein of interest
2. Antibody labelled/conjugated with ‘reporter’- normally fluorescent
Advantages of direct immunohistochemistry
More specific- only binding to one antigen
Involves fewer steps
Useful when antigen is abundant
Disadvantages of direct immunohistochemistry
Expensive- requires access to different specific Abs
Label may interfere with Ab binding to epitope
Describe indirect immunohistochemistry
- Buy antibody specific for protein of interest
- Primary antibody binds to antigen
- Secondary antibody raised against own Ig from different species and labelled
Advantages of indirect immunohistochemistry
More sensitive
Signal amplification
Cheaper
Useful when antigen is not abundant
State the immunostaining process
- Block
- Wash with PBS-Tween
- Incubate 1*
- Wash with PBS-Tween
- Incubate 2*
- Wash with PBS-Tween
- Visualise signal (conjugate enzyme) via colour or fluorophore
What is a ‘block’ in immunostaining
prevents non-specific proteins from binding
What sample type does Western blotting require
Mix of proteins in a tube
State the steps of Western Blotting
- Take cells from sample
- Homogenise cells (mash them up) or Lyse cells
- Run SDS (detergent) Page gel separate proteins by size, not charge
- Transfer/blot cells
- Wash, Add primary antibody
- Wash, Add secondary antibody with stain*
- Visualise results
*can do direct/indirect like immunohistochemistry
Purpose of PCR?
to amplify target DNA
What are the steps involved in PCR
- Add buffer and Double stranded DNA template
- Add dNTPs
- Design primer- short nucleotide primer complementary to your gene of interest
- Run PCR ~30 times
- Denaturing stage 95*C
- Annealing stage 55*C
- Add specific primers
- Extending stage 72*C
- Add Taq polymerase (heat stable)
- Run PCR on agarose gel electrophoresis
- Visualise e.g. SYBR-Green
What are the steps of RT-PCR
- Isolate RNA from sample
- Convert to DNA using reverse transcriptase
- Denature at 98*C
- Anneal at 55*C
- Add specific primers
- Extend at 72*C
- Taq polymerase extends primers adding nucleotides
- Cycle repeats to amplify DNA
- Visualise the signal
What does RT-PCR require that normal PCR doesn’t and why?
Reverse Transcriptase
To convert RNA to DNA
What is the purpose of ELISA
Uses specific antibodies/antigen to detect and quantify a particular protein or molecule
Explain the steps of ELISA
- Wells are coated in antigen of interest
- Wells filled with dilutions of the patient’s serum
- If antibodies against the antigen are present, they will bind to the antigen fixed to the bottom of the wells
- Only antigen-specific antibodies will bind to the wells
- Wells are washed out to remove all unbound antibodies
- Animal antibodies against the human antibodies is added
- These antibodies are covalently conjugated to an enzyme
- The wells are then washed again, to remove any unbound enzyme-conjugated antibody
- Solution of colorogenic enzyme substrate is added
- The interaction of the substrate with the enzyme on the second antibody generates visible colour
- Development of colour in the wells can be seen with the naked eye, or electronic plate reader
What is flow cytometry
A technology used to analyse the physical and chemical characteristics of particles in a fluid as it passes through at least one laser.
What does flow cytometry measure
fluorescence intensity from fluorescent-labelled antibodies, against specific proteins or antigens.
What procedures are flow cytometry used to perform
Cell counting
• Cell sorting
• Detection of biomarkers
• Protein engineering
Explain the steps in flow cytometry
- sample added
- hydrodynamic focusing as cells pass through in single file
- fluorescence emitted from stained cells
- scattered light by cells detected
What is Western Blotting
Use of a specific antibody to detect and quantify a particular protein in a sample
