measurements of proteins Flashcards
what is spectrophotometry?
- measurement of the absorption of electromagnetic radiation of a substance at different wavelengths of the spectrum
how does spectrophotometry work?
- 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
what are nanodrops? what is used for?
- advancement which uses spectrophotometry to quantify nucleic acids
- commonly used to check the purity of isolated nucleic acid
what wavelength do nucleic acid absorb light?
- absorb ultraviolet light at a wavelength of 260nm
what is spectrophotometry used to measure?
- total protein abundance
what is the Bradford Assay used for?
- used to standardise the amount of protein that they are analysing
how does the Bradford Assay work?
- protein mixed with a dye called Coomassie blue
- in acidic conditions this results in a colour change brown- blue
when can protein quantity be detected?
- at 595nm
what is the polymerase chain reaction?
- technique that allows you to make many copies of a particular gene
how does PCR work?
- reverse transcriptase plus the addition of nucleotides form complimentary DNA
- heat can split complementary DNA
- DNA polymerase amplifies the quantity of DNA
what is real time quantitative PCR?
- PCR based technique that couples amplification of a target DNA sequence with quantification of concentration of that DNA species
how can you measure specific proteins with antibodies?
- 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
what is the western blot?
- widely used to detect protein in a tissue homogenate/ protein extract
how does western blot work?
- sample collection
- protein extraction (homogenisation)
- electrophoresis to separate proteins according to size
- electrotransfering protein from gel to membrane
- antibody probing and detection
what does ELISA test stand for? what is it?
- enzyme linked immunosorbent assay
- blood test used to measure proteins in bodily fluid in order to quantify the proteins
how does the ELISA test work?
- antibodies bind to the specific protein of interest and enzyme- linked secondary antibody is used to produce a measurable signal
how can skeletal muscle contraction be replicated without nervous system?
- 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
what is used to stimulate muscle contraction?
- repeated electrical pulse stimulation (EPS) which allows the release of calcium ions from the sarcoplasmic reticulum to enable the machinery of actin and myosin
what does every pulse cause? what does rapid pulse cause?
- 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
what is an example of manipulation of what happens in muscles?
- verapamil blocks Ca2+ channels
what is type of technique is EPS?
- looks like muscle contraction
- mediated by calcium ions
how do you measure lactate production by myocytes?
- 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
what is lactate production equation and what enzyme catalyses this?
lactate + NAD = pyruvate + NADH + H+
- catalysed by lactate dehydrogenase
what is PGC1 alpha mRNA?
- transcription factor that plays a prominent role in adaptations of exercise
how can PCG1 alpha mRNA be measured in myotubes?
- polymerase chain reaction
- take the cells and isolate the mRNA so RNA can be split open and provided with nucleotides and enzyme reverse transcriptase
what is needed to amplify the amount of RNA?
- 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
how can GDF15 protein secretion be measured?
- via ELISA test; need a specific antibody that is complementary to GDF15
write the steps involved in measuring GDF15 protein secretion
- 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
what happens after you add the sample to the microplate when measuring GDF15 protein secretion?
- 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
how can myosin heavy chain 1 protein expression be measured?
- 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
what is immunohistochemistry?
- 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
what happens after the thin cross section is incubated with antibodies?
- 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
