Part 3 Slides 42-91 Flashcards

Question

What signifies that a column is charge with nickle

Answer 1

Blue colour

Answer 2

The bead has either NTA or IDA (nitrilotriacetic acid, iminodiacetic acid) as a chela for for metal ion Then NTA is charged with the metal (ni) to make Ni-NTA Then the nitrogen on the his tag interacts with the metal

Answer 3

The tags bind best to the column at ph 7.5 and 150 mM ionic strength (physiological) Wash buffer has low amount of imidazole to prevent non specific binding of protiens that naturally have his, by washing them out Also salt and low concentration detergent to stop nonspecific binding Elute with high imidazole

Answer 4

Since high salt and chaotropic agents like 8M urea and 6M guandiunium are compatible with the column You can use them to first denature the protien to get is soluble then do IMAC

Answer 5

The protiens that are insoluble and aggreagate we use denaturants to solubilize them to purify in IMAC After being purified, they refold and precipitate after the urea is removed

Answer 6

Gst and MBP needs the protien to be properly folded and have the 3D binding site to be functional so it can bind to the matrix But IMAC doesn’t need this since his tags aren’t 3D, which is why we can purify inclusion bodies using denaturants

Answer 7

Reducing agents and chelators (EDTA), so can’t use them to denature the inclusion bodies

Answer 8

To make antibody’s To use in far western blots also called overlays (since western blots can use both soluble and insoluble protiens)

Answer 9

The DNA binding to DNA on a membrane (southern blot) was done by a man named southern, they kept the theme

Answer 10

Western blot: have a protien on the membrane with an epitope, primary antibody binds to this epitope, secondary antibody for labelling binds to primary Far western: another protien bind to epitope instead of antibody. Now a protien protien interaction then the antibody binds the other protien

Answer 11

In western blot have anti EGFR, see high expression of EGFR band in certain patients In far western blot: had GST-SH2 PI3K, the SH2 domain in PI3K docks to the EGFR since the EGFR has phosphorylated tyrosine (protien protien interaction) The extra band in far western could be: The band in far western is actually a diff protien with phos tyrosines the EGFR protien is cleaved, so epitope gone but still sh2 binding to phos tyrosine, causes band in far western and none in western

Answer 12

Peptide AC: peptide bound to the matrix Co IP: antibody bound to agarose bead, get protien plus binding partners Small molecule AC: coupled microcystin to the matrix to purify PP1, couple ATP to the matrix Pulldowns Displacement AC Tap-tag (tandem affinity chromatography)

Answer 13

There is a peptide on a bead that is phos and dephos (has RVSF phos and no phos) Get diff protiens bound to this peptide in phos and de phos form Ex. PP1 binds and elutes with de phos version but not to phos version, phosphorylation prevent binding of pp1

Answer 14

One band with 55 and one at 25 (heavy and light chain) and the antigen of the antibody that was bound to the protien A resin To remove the chains in the SDs page , covalently couple the antibody to protien A to just get the antigen and no antibody

Answer 15

Used kif18a antibody on the matrix to bind kif18a and eluted Did western blot with anti pp1 and anti kif18a, saw that pp1 was also in that eluted sample meaning it’s interacting with kif18a Then did pull down assay with his tagged kif18a and saw that pp1 gets pulled down

Answer 16

Have a pure and tagged protien (bait) that pulls down its binding partner (prey) For example, doing IMAC, his tagging kif18a and seeing if PP1 comes out with the kif18a

Answer 17

Put each tube through ni-NTA column Tube 1: no pp1, his kif18a or RARA kif18a , no bands Tube 2: just pp1, control since could be binding non specifically, no bands Tube 3: pp1 and his kif18a, saw pp1 band and kof18 band meaning pp1 pulled down with kif18a Tube 4: pp1 and RARA kif18a, control, no pp1 band but kif18a band, show that if no RVSF motif PP1 doesn’t bind So showed that pp1 binds to k18a directly through the RVSF motif

Answer 18

Using a peptide to displace specific binding partners For example want pp1 binding partners but not pp1 Use a peptide with RVRW motif to bind to pp1 and displace binding partners in elution

Answer 19

VERY BAD TOXIN A whole range of organisms create this small molecule/toxin that binds to enzymes to inhibit their function Sweden releases raw sewage to the rivers, nitrogenous compounds in the sewage let’s the blue green algae to thrive and make Microcystin Pollution make let’s microcystin in the water and kill us

Answer 20

The RVRW peptide being able to elute kif18a from the matrix (displacement chromatography) shows that kif18a binds to pp1 directly using a RVRW motif

Answer 21

1. The displacement chromatography with pp1 on the MC column, gave kif18a and K9 as binding partners 2. The co-immunoprecipitation of kif18a, gave kif18 and pp1 in elution 3. The pull down assay of pp1 with kif18a, showed pp1 binds kif18a through the motif RVRW since didn’t get pulled down with mutated RARA

Answer 22

It’s a Kinesin (motor protein) that binds pp1 directly in the nucleus to take it to the mitotic spindle kinetochore for a de phosphorylation event Found by: Cloned kif18a with RFP, and have the PP1 with GFP, Had a stain that recognizes the dna Saw that kif18a and pp1 showed up at the end of the microtubules which were binding to the chromosomes

Answer 23

Has easy genetics so we do large scale experiments with it We can do homologous recombination to it where we have a tagged protien and expresses that tagged version in the yeast in place of the WT

Answer 24

Tandem affinity purification (two affinity chromatography in a row) You fuse the TAP tag genome to the target protien genome and use homologous recombination to replace the endogenous WT protien with the TAP tagged one Do this because if the endogenous protien is being overexpressed, it accosiates with non-natural binding partners So maintain the normal levels by just completely replacing the endogenous protien with the tagged one through homologous recombination

Answer 25

They are basically a map of different protien protien interactions Ex. Can map that pp1 interacts with kif18a

Answer 26

The tag has two copties or the protien A igG binding domains and a calmodulin binding peptide (CBP) Separated by a TEV protease cleavage site Protien- CBP- protease site-A-A Can be flipped so N-C term is protien then protons A Or N-C is protien A then protien When ca binds the CAM, the CAM has a conformational change with its binding site exposed and binds to the CBP

Answer 27

Because we’re looking for binding partners, the protien could bind at the n term or the c term In each way either the n term or the c term of the protien is covered by the CAM binding protein So we do this to make sure all binding sites can be exposed Ex. N-protein CAM- C (n term exposed with protien at the start) N - CAM-protein -C (c term exposed with protien at the end)

Answer 28

you’ve expressed the protien with the tag it, has its normal binding partners, expressed at normal levels First affinity step: Put the cell lysate onto an igG matrix, non working end binds the protien A with high affinity While bound to matrix, use the TEV to cleave Now binding partners-protien-CBP elutes and protease, leaving non specific binders on the bead Second affinity step: The eluate binds to CAM coated beads with calcium in the buffer So now CAM on the bead has conf change, bind the CBP-protien-binding partners Wash to remove contaminants Use egta to chelate the ca ions to make CAM change conf to close Elutes the pure CBP-protien-binding partners All nonspecific binders gone because using egta to elute

Answer 29

Ran 4 lanes with snu71p WT and TAP tagged Just using cam bead to purify: tap tagged showed band of the snu71p protein (with proteins A and CBP) and some contaminants, WT showed all contaminants and non specific binding Using just igG with TEV protease: tap tagged showed band of the cleaved snu71p protein (with just CBP), some contaminants, and the dirty TEV protease , WT showed all contaminants and non specific binding and tag contaminants Just TEV protease: Control , Showed that the protease is very dirty and contributes to some of the bands we see in the experiment Tandem regular tap tag method: BEST RESULTS tap tagged showed band of the snu71p protein and binding partners and barely any contaminants, WT no contaminants since all washed out

Answer 30

By seeing that each band gets fainter as it goes down, for every molecule of sun there one molecule of binding partner and one mocleule of another binding partner Coosmasie binds the surface of the protiens, if longer protien more intense band 1:1 binding

Answer 31

Wanted to see if protien kinase inhibitors were specific or not by finding the targets of 243 kinase inhibitors Did this by using kinobeads and using MS to see if the inhibitor were specific to what they were made for

Answer 32

They let us purify endogenous protein kinases (no non protien kinases like pyruvate kinase) By using quantitative mass spec, let us do label free measurements (don’t have to buys expensive reagent) of the interaction of the kinase with a bunch of protiens in parallel

Answer 33

TMT labelling (chemically label the peptides and quantify them using that label) Label free

Answer 34

Since protien kinases are disregulated in diseases like cancer, we use these small protein kinase inhibitors to target and stop them Kinases are control in many of the cellular processes so if diregulated they cause big effects

Answer 35

Crizotinib and imatinib (gleevac) They are tyrosine kinase inhibitors that aren’t as specific as we thought they were have off target/side effects Need the drug to be a specific as possible to remove off target/side effects

Answer 36

It’s a phylogenetic tree showing the relationship of all kinases to other kinases based on their sequence Branches show ones that are closer to each other in the tree and are more closely related to each other than the other kinases The circles mark protien kinases that the drug (imatinib or crizotinib) bind to in the human kinome The bigger the circle the tighter the binding of the drug to the kinase Shows that the drugs bind to many different protien kinases

Answer 37

Most protien get phosphorylated, 75% Ser (86%) thr (12%) tyr (2%)

Answer 38

Use atp to phosphorylate specific residues on specific protiens (not random process) Add phosphryl group

Answer 39

The 518 PK in the human genome GPCR are first

Answer 40

The atp binding pocket of the protien kinase The substrate and atp has to dock in the kinase Drugs are trying to be specific to certain kinases to inhibit them, but each protien kinase binds atp in the same way, hard to make the drugs specific if they all use the same way to bind atp

Answer 41

On beads link 5 known protien kinase inhibitors that are non specific and bind all kinds of kinases Mixed the beadS together Run lysate of 4 different cell lines: want this because they express a bunch of diff kinases that can be extracted Allow them to pull out all the protien kinases in the cell (missing some but may be because they weren’t expressed in the cell) Do dose dependent competition with the small molecule inhibitors Using LC-MS/MS Sequenced the peptides of the collected kinases to see which protein kinases were left on the beads after the dose dependent competition

Answer 42

Have varied amount of small molecule drug competing for binding the kinase with the kino bead Orange green yellow kinase Have zero small molecule inhibitor drug, do label free lc-MS/MS on the bead, see that the protien kinases dock the kino bead and high amounts of each kinase are seen in ms/ms since drug didn’t displace them When adding inhibitor, with add it to the cell extract to it binds to the kinases, then run across the beads. Any kinase the drug binds to will not bind to the kinobead (since active site occupied by the inhibitor) and barely show up in MS/MS One that decreases most would mean drug bind to it with high affinity Keep increasing the concentrations in the cell extracts, the MS/MS of the high concentration inhibitor would show less signal of the kinase the drug has high affinity for same amount of yellow protien in zero as in high shows that the inhibitor doesn’t bind to that kinase

Answer 43

Range of 3 nM to 30,000 nM

Answer 44

Found that fausidil targets PRKX, not Rock 1 and rock 2 Hydroxyfausidil targets ROCK1 and ROCK2

Answer 45

Did the kinobeads competitive pull-down assay To test Fausidil: The binding curve showed that as they increased the amount of Fasudil, the binding or PRKX to the column decreased more than ROCK1 and 2 The activity curve shows activity of PKRX decrease more than ROCK1 and ROCK2, meaning more of it is inactivated by Fasudil, more sensitive to the drug Then did same but with hydroxyfasudil Binding curve showed that PRKX not sensitive to drug at all , but ROCK1 and 2 decrease even more now Same in the activity, ROCK1 and ROCK2 decrease

Answer 46

250 out of the 518 in the cell

Answer 47

Most KI drugs target more than one protien and can’t be extremely specific So we study their actual targets to explain undesired side effects and to see if we can accept this degree of side effects To repurpose the drug (might find it binds to another type of kinase to stop a diff disease)

Answer 48

Found that some drugs show exquisite selectivity but others bound more that 100 kinases simultaneously Meaning it’s hard to make specific kinase inhibitor drugs using those inhibitors Repurposing: Also found new targets for already made drugs , like SIK2 inhibitor targets TNF alpha and IL-10 production

Answer 49

Kinases that are inactive but still expressed in cells

Answer 50

They found that many drugs target things other than their expected target For example they target the kinase PDXK which phosphorylates vitamin B6 to make it, can lead to side effects

Answer 51

Each black circle is a kinase inhibitor drug The size of the circle is how many kinases it binds to (bigger circle, bind more kinases) For example the drug ponatinib which is a tyrosine kinase inhibitor, is supposed to target a kinase for chronic myeloid leukaemia But it binds to 39 other PK and has side effects So bigger black circle

Part 3 Slides 42-91 Flashcards

(76 cards)