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Disease, Diagnostics and Therapeutics > Specific Laboratory Techniques > Flashcards

Specific Laboratory Techniques Flashcards

1
Q

What sample type does immunohistochemistry chemistry require?

A
  • Very thin section of tissue (frozen or embedded in paraffin).
  • Antigens visible on tissue slice
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2
Q

Describe direct immunohistochemistry

A
  1. Buy antibody specific for protein of interest

2. Antibody labelled/conjugated with ‘reporter’- normally fluorescent

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3
Q

Advantages of direct immunohistochemistry

A

More specific- only binding to one antigen

Involves fewer steps

Useful when antigen is abundant

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4
Q

Disadvantages of direct immunohistochemistry

A

Expensive- requires access to different specific Abs

Label may interfere with Ab binding to epitope

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5
Q

Describe indirect immunohistochemistry

A
  1. Buy antibody specific for protein of interest
  2. Primary antibody binds to antigen
  3. Secondary antibody raised against own Ig from different species and labelled
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6
Q

Advantages of indirect immunohistochemistry

A

More sensitive
Signal amplification
Cheaper
Useful when antigen is not abundant

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7
Q

State the immunostaining process

A
  1. Block
  2. Wash with PBS-Tween
  3. Incubate 1*
  4. Wash with PBS-Tween
  5. Incubate 2*
  6. Wash with PBS-Tween
  7. Visualise signal (conjugate enzyme) via colour or fluorophore
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8
Q

What is a ‘block’ in immunostaining

A

prevents non-specific proteins from binding

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9
Q

What sample type does Western blotting require

A

Mix of proteins in a tube

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10
Q

State the steps of Western Blotting

A
  1. Take cells from sample
  2. Homogenise cells (mash them up) or Lyse cells
  3. Run SDS (detergent) Page gel separate proteins by size, not charge
  4. Transfer/blot cells
  5. Wash, Add primary antibody
  6. Wash, Add secondary antibody with stain*
  7. Visualise results

*can do direct/indirect like immunohistochemistry

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11
Q

Purpose of PCR?

A

to amplify target DNA

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12
Q

What are the steps involved in PCR

A
  1. Add buffer and Double stranded DNA template
  2. Add dNTPs
  3. Design primer- short nucleotide primer complementary to your gene of interest
  4. Run PCR ~30 times
  5. Denaturing stage 95*C
  6. Annealing stage 55*C
  7. Add specific primers
  8. Extending stage 72*C
  9. Add Taq polymerase (heat stable)
  10. Run PCR on agarose gel electrophoresis
  11. Visualise e.g. SYBR-Green
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13
Q

What are the steps of RT-PCR

A
  1. Isolate RNA from sample
  2. Convert to DNA using reverse transcriptase
  3. Denature at 98*C
  4. Anneal at 55*C
  5. Add specific primers
  6. Extend at 72*C
  7. Taq polymerase extends primers adding nucleotides
  8. Cycle repeats to amplify DNA
  9. Visualise the signal
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14
Q

What does RT-PCR require that normal PCR doesn’t and why?

A

Reverse Transcriptase

To convert RNA to DNA

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15
Q

What is the purpose of ELISA

A

Uses specific antibodies/antigen to detect and quantify a particular protein or molecule

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16
Q

Explain the steps of ELISA

A
  1. Wells are coated in antigen of interest
  2. Wells filled with dilutions of the patient’s serum
  3. If antibodies against the antigen are present, they will bind to the antigen fixed to the bottom of the wells
  4. Only antigen-specific antibodies will bind to the wells
  5. Wells are washed out to remove all unbound antibodies
  6. Animal antibodies against the human antibodies is added
  7. These antibodies are covalently conjugated to an enzyme
  8. The wells are then washed again, to remove any unbound enzyme-conjugated antibody
  9. Solution of colorogenic enzyme substrate is added
  10. The interaction of the substrate with the enzyme on the second antibody generates visible colour
  11. Development of colour in the wells can be seen with the naked eye, or electronic plate reader
17
Q

What is flow cytometry

A

A technology used to analyse the physical and chemical characteristics of particles in a fluid as it passes through at least one laser.

18
Q

What does flow cytometry measure

A

fluorescence intensity from fluorescent-labelled antibodies, against specific proteins or antigens.

19
Q

What procedures are flow cytometry used to perform

A

Cell counting
• Cell sorting
• Detection of biomarkers
• Protein engineering

20
Q

Explain the steps in flow cytometry

A
  1. sample added
  2. hydrodynamic focusing as cells pass through in single file
  3. fluorescence emitted from stained cells
  4. scattered light by cells detected
21
Q

What is Western Blotting

A

Use of a specific antibody to detect and quantify a particular protein in a sample

Disease, Diagnostics and Therapeutics (14 decks)

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