Data Handling Definitions Flashcards
Ni-affinity column
Apply protein solution to Ni-affinity column. Add low imidazole buffer solution. his-tag will stick to the column. Wash to remove weaker binding proteins. Elute with high imidazole buffer solution. Remove His-tag by adding Thrombin, then purify to leave just protein
Radioactive p32 detection
Add cAMP dependant kinase catalytic domain from heart muscle. Add 32ATP and allow reaction to occur. 32P will be incorporated into the Ser residue of the kinase site through phosphorylation. Run proteins on gel and detect radioactivity with Densitrometry Imaging
rp-HPLC
Reverse phase High Performance Liquid Chromatography . Uses a non-polar stationary phase and polar mobile phase. Separates fragments based on hydrophobic interactions with the stationary phase - more complex molecules interact stronger with stationary and are retained for longer = longer retention time
Gram (+) vs Gram (-) PG
Gram (+) has Glutamine at 2, Lysine at 3
Gram (-) has mDAP
What does the first HPLC peak represent? What is its most likely function
Represents the simplest fragment = most abundant monomer
Usually the most abundant monomer acts as the acceptor in crosslinking
Western Blot
Comparing protein abundance - separates on a gel
Immunoprecipitation
Isolates and purifies protein from a mix using antibodies - protein analysed using western blot etc…
What does p value tell us
How likely it is we got the results we did if the null hypothesis is correct
What gel do we need to use to separate small proteins
One with higher acrylamide percentage to slow the proteins down
What is Gaussian distribution
tells us the data at 60% percent of the bell curve is 1SD away each side
Variables
raw data values from the expreriment
Parameters
Values that are adjusted during data fitting to fit a model curve to the test results
Constants
Fixed values of the exam conditions.
What are the steps of PG purification?
Extraction = the bacteria is grown till exponential phase and then boiled in 4% SDS for 30 minutes -> washed with distilled water
Purification = treated with a protease (pronase, or trpsin) -> washed -> freeze-dry -> treated with HF or HCl to remove Teichoic acids, capsules, and other PG associated molecules
PG digestion = The extracted sample is broken down by enzymes or lysozomes -> breaking down the PG into soluble fragments (by cleaving the MurNAc – GlcNAc bonds) -> this producing monomers, dimers, trimers, etc.
Reduction = As the monomers are sugars, they have reducing end at position 1 (an OH group). Using sodium borohydride, this attacks the molecule by nucleophilic attack, locking the molecule into a single conformation, with nowhere for the OH’s free electron to substitute, this prevents multiple peaks from forming,
