measurements of proteins

Question 1

what is spectrophotometry?

Answer 1

• measurement of the absorption of electromagnetic radiation of a substance at different wavelengths of the spectrum

Question 2

how does spectrophotometry work?

Answer 2

• many compounds in a solution absorb visible light in proportion to their concentration
• while many biomolecules are colourless combining with a reagent produces a coloured compound

Question 3

what are nanodrops? what is used for?

Answer 3

• advancement which uses spectrophotometry to quantify nucleic acids
• commonly used to check the purity of isolated nucleic acid

Question 4

what wavelength do nucleic acid absorb light?

Answer 4

• absorb ultraviolet light at a wavelength of 260nm

Question 5

what is spectrophotometry used to measure?

Answer 5

• total protein abundance

Question 6

what is the Bradford Assay used for?

Answer 6

• used to standardise the amount of protein that they are analysing

Question 7

how does the Bradford Assay work?

Answer 7

• protein mixed with a dye called Coomassie blue
• in acidic conditions this results in a colour change brown- blue

Question 8

when can protein quantity be detected?

Answer 8

• at 595nm

Question 9

what is the polymerase chain reaction?

Answer 9

• technique that allows you to make many copies of a particular gene

Question 10

how does PCR work?

Answer 10

• reverse transcriptase plus the addition of nucleotides form complimentary DNA
• heat can split complementary DNA
• DNA polymerase amplifies the quantity of DNA

Question 11

what is real time quantitative PCR?

Answer 11

• PCR based technique that couples amplification of a target DNA sequence with quantification of concentration of that DNA species

Question 12

how can you measure specific proteins with antibodies?

Answer 12

• antigen injected into host species, usually a rabbit
• rabbit then produces antibodies in response to the protein of interest
• can then be used to detect and quantify the protein in various assays

Question 13

what is the western blot?

Answer 13

• widely used to detect protein in a tissue homogenate/ protein extract

Question 14

how does western blot work?

Answer 14

• sample collection
• protein extraction (homogenisation)
• electrophoresis to separate proteins according to size
• electrotransfering protein from gel to membrane
• antibody probing and detection

Question 15

what does ELISA test stand for? what is it?

Answer 15

• enzyme linked immunosorbent assay
• blood test used to measure proteins in bodily fluid in order to quantify the proteins

Question 16

how does the ELISA test work?

Answer 16

• antibodies bind to the specific protein of interest and enzyme- linked secondary antibody is used to produce a measurable signal

Question 17

how can skeletal muscle contraction be replicated without nervous system?

Answer 17

• grow cells in labs and provide them with media (nutrients needed for growth)
• myocytes treated in a way to form multinucleated myotubes
• myoblasts manipulated to make them look like muscles

Question 18

what is used to stimulate muscle contraction?

Answer 18

• repeated electrical pulse stimulation (EPS) which allows the release of calcium ions from the sarcoplasmic reticulum to enable the machinery of actin and myosin

Question 19

what does every pulse cause? what does rapid pulse cause?

Answer 19

• release of calcium ions
• rapid pulses cause a fused tetanus so contraction happens all the time
• need energy for Ca2+ to get back to SR to allow relaxation

Question 20

what is an example of manipulation of what happens in muscles?

Answer 20

• verapamil blocks Ca2+ channels

Question 21

what is type of technique is EPS?

Answer 21

• looks like muscle contraction
• mediated by calcium ions

Question 22

how do you measure lactate production by myocytes?

Answer 22

• NAD+/ NADH linked enzymatic assay
• measured using spectrophotometry ; a lot of assays are linked to reduction/ oxidation molecules
• oxidised form NAD+ can accept a hydrogen from an oxidised metabolite forming NAD

Question 23

what is lactate production equation and what enzyme catalyses this?

Answer 23

lactate + NAD = pyruvate + NADH + H+
- catalysed by lactate dehydrogenase

Question 24

what is PGC1 alpha mRNA?

Answer 24

• transcription factor that plays a prominent role in adaptations of exercise

Question 25

how can PCG1 alpha mRNA be measured in myotubes?

Answer 25

• polymerase chain reaction
• take the cells and isolate the mRNA so RNA can be split open and provided with nucleotides and enzyme reverse transcriptase

Question 26

what is needed to amplify the amount of RNA?

Answer 26

• specific primers can be used to amplify the amount of RNA in order to make more DNA via DNA polymerase
• if lactate increases then PCG1a also increases

Question 27

how can GDF15 protein secretion be measured?

Answer 27

• via ELISA test; need a specific antibody that is complementary to GDF15

Question 28

write the steps involved in measuring GDF15 protein secretion

Answer 28

• inject antigen into host as it will respond by making antibodies
• purify antibodies and use them to bind a microplate
• add sample then wash microplate

Question 29

what happens after you add the sample to the microplate when measuring GDF15 protein secretion?

Answer 29

• add detection antibody (has conjugate tagged to it so that when substrate is added it changes colour)
• wash plate, add substrate and then read the microplate
• colour change directly related to amount of GDF15

Question 30

how can myosin heavy chain 1 protein expression be measured?

Answer 30

• via western blotting
• extract the cells, homogenise the tissues, run the proteins through a gel via electrophoresis
• transfer protein to a membrane and add antibody specific to antigen

Question 31

what is immunohistochemistry?

Answer 31

• muscle biopsy is a cryo- sectioned to produce a very thin cross section of the muscle
• incubated with antibodies against main myosin heavy chain isoforms

Question 32

what happens after the thin cross section is incubated with antibodies?

Answer 32

• secondary antibody is added which contains a fluorescent signal to show different types of fibres
• relative contribution of each fibre can determine what exercise your most suited to