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YEAR 2: Skeletal Muscle in Health and Disease (MGEM2017 UNUK) (SPR1 23-24) > molecular methods in Research > Flashcards

molecular methods in Research Flashcards

1
Q

what is a post-translational modification (PTM) on a protein?

A

post-translational modification which would change the way a protein works

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2
Q

what are 3 forms of tissue homogenisation for protein extraction

A
  • polytron
  • using scissors to chop them up
  • sonication
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3
Q

what are the 5 steps of western blotting?

A
  1. Protein extraction
  2. SDS-polyacrylamide Gel electrophoresis
  3. blocking and electro-blotting
  4. antibody conjugation/immunoprecipitation and Sourcing

5.membrane stripping

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4
Q

outline how SDS-Polyacrylamide Gel Electrophoresis works in 4 steps

A
  1. complete Protein extraction and add disulphide bridges
  2. load equal protein samples in gel wells
  3. then apply positive electrical charge away from the proteins
  4. over time, the smaller molecules move more quickly throughout the gel and the larger protein molecules will move more slowly throughout the gel.
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5
Q

what 2 reasons make it important to separate out proteins by molecular weight using gel electrophesis?

A
  • as you are looking from a pool of 30,000+ proteins, it can get messy if you don’t separate them out to know exact proteins
  • if you know the predicted molecular weight, based on amino-acid sequence, you can vet for errors in the gel electrophoresis or protein size if you have incorrect separation
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6
Q

blocking and electro-blotting

what is electro-blotting?

A

applying an electrical charge to move proteins separated by gel electrophoresis from initial membrane to another

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7
Q

what 2 compounds can be used for electro-blotting?

A
  • PVDF

OR

  • nitocellulose
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8
Q

what are monoclonal antibodies

A

antibodies produced by one tyep of immune cell and are all clones of a single parent cell

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9
Q

what are polyclonal antibodies?

A

mixture of immunoglobulin molecules secreted against a specific antigen

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10
Q

why do you use a secondary antibody in antibody conjugation and Source?

A

to allow for amplification of binding, a the secondary antibody will bind all on the primary antibodies

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11
Q

what is a feature of phospho-specific antibodies?

A

they only recognise epitoeps when phosphorylated at specific residues

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12
Q

what is stripping the membrane after antibody conjugation and Source?

A

removal of antibodies from the membrane using either pH, heat or detergent

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13
Q

what are three general considerations when using softwares to quantify your protein extraction

A
  • beware of pixel saturation
  • use a loading control/house-keeping protein like GAPDH or actin to confer mRNA stability with treatment and between subjects
  • use Ponceau S to stain all proteins on the membrane to show equal loading
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14
Q

what is immunoprecipitation

A

technique of precipitating a protein antigen out of solution by using an antibody that specifically binds to a particular protein

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15
Q

outline in 5 steps how immunoprecipitation works

A
  1. add antibody that works against protein of interest
  2. antibody binds to protein of interest
  3. adding protein A or G makes antibody-protein complexes insoluble
  4. centrifugation of solution pellets the complexes.
  5. The resultant supernatant is removed and is washed off
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16
Q

where is protein A derived from

A

staphylococcus aureus

17
Q

where is protein G derived from?

A

b-hemolytic Streptococci

18
Q

what does it mean if your target protein showed a 10x fold change increase compared to your starting, control protein but your loading control protein (actin, GAPDH etc) showed a 2x fold change increase compared to the starting control protein

A

it means that the NET fold increase is 5x as the starting control protein amount wasn’t accurately quantified

( corrected fold increase = 10/2 = 5 )

19
Q

what is the equation that links ratio target gene in experimental/control, fold change in target gene and fold change in reference gene

A

ratio target gene in experimental/control = fold change in target gene / fold change in reference gene

/ = divided by in second half of equation

20
Q

what are 4 methods of measuring gene expression/mRNA Abundance?

A
  • reverse transcriptase polymerase chain reaction (RT-PCR)
  • traditional thermal cycling
  • northern blotting
  • Microarray technology
21
Q

what is the goal of measuring gene expression/mRNA abundance?

A

goal is to measure effects of interventions (i.e exercise) on the expression of a gene (AKA its transcription)

22
Q

what are 3 benefits of real-time PCR

A
  • excellent sensitivity
  • high versatility
  • simiplicity
23
Q

state the 5 steps of RT-PCR

A
  1. get tissue
  2. extract RNA
  3. copy into cDNA using reverse transcriptase
  4. do Real time-PCR
  5. analyze results
24
Q

what is the more prodominant RNA species

A

ribosomal RNA

25
Q

outline how RNA extraction works in 3 steps

A
  1. Use Trizol to extract Proteins, DNA and RNA from tissue sample. Trizol will dissolve the proteins, DNA and RNA for this to work.
  2. Use Chloroform to separate out the organic matter from the inorganic matter (separate the DNA from the RNA)
  3. use isopropyl alcohol to precipitate the RNA
26
Q

what 3 things should the RNA precipitate be free of in RNA extraction?

A

should be free of:

  • proteins
  • gDNA
  • PCR inhibitors
27
Q

why can’t you just quantify and compare the amounts of RNA in different samples and what do you do to overcome this?

A
  • there isn’t enough RNA material to do that
  • overcome this by amplifying the RNA
28
Q

outline in 4 steps how reverse transcription works in RT-PCR

A
  1. Have template RNA
  2. Anneal RNA and then add primers
  3. add reverse transcriptase enzyme to synthesis complementary DNA fragments
  4. Denature the cDNA-RNA complex, so you only have single stranded cDNA
29
Q

polymerase chain reaction

what 4 things are used in PCR?

A

- DNA polymerase enzyme
- fluoresecent dye such as SYBR green
- Deoxygribonucleotides (dNTPs)
- Gene specific primers (derived bio-informatically)

30
Q

what is the purpose of transferring from gel to membrane after SDS-PAGE via electro-blotting

A
  • membrane has good affinity for antibodies
  • gel is impenetrable to antibodies
31
Q

Outline how SDS is added to the protein sample molecules in SDS PAGE and how gel electrophoresis works after this

A
  1. Mercaptoethanol denatures protein
  2. SDS binds protein stoichiometrically
  3. SDS is now bound to the protein sample

YEAR 2: Skeletal Muscle in Health and Disease (MGEM2017 UNUK) (SPR1 23-24) (23 decks)

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