Immunoprecipitation (Stukenberg lab) and Small Molecules for Xenopus Research: Difference between pages

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Protocol submitted by VGP from Stukenberg lab protocols [http://www.xenbase.org/community/person.do?method=display&personId=1311&tabId=0]
==Notice to our users==
Please help us to improve this small molecules wiki and our other wikis by registering for a wiki account through our normal feedback email.


==Important small molecule references==
*[http://www.xenbase.org/literature/article.do?method=display&articleId=35021 Database of Small Molecules by Feldman and Hogue 2006] at [https://pubchem.ncbi.nlm.nih.gov/ PubChem]
*[http://www.xenbase.org/literature/article.do?method=display&articleId=47903 Wnt/Notch by Myers and Krieg 2014]
*[http://www.xenbase.org/literature/article.do?method=display&articleId=970 Ubiquitin-proteasome by Salic and King 2005]


link to protocol page [http://people.virginia.edu/~djb6t/LabWeb/frogs.htm]
==Alphabetical List of Small Molecules With Literature References==


*[[1,3-Dicyclohexylurea]]
*[[11-cis retinal]]
*[[18alpha-glycyrretinic acid]]
*[[2'-deoxyadenosine]]
*[[2-APB]]
*[[3,3,5-triiodothyronine (T3)]]
*[[5HT (seratonin)]]
*[[8-Pcpt-2-O-Me-cAMP-AM]]
*[[activin]]
*[[AG1296]]
*[[AG1879]]
*[[AGN193109]]
*[[alpha-amanitin]]
*[[anandamide]]
*[[apelin]]
*[[aphidicolin]]
*[[AZD1480]]
*[[Bafilomycin A1]]
*[[BAPTA-AM]]
*[[Barasertib]]
*[[Benzaldehyde, 3-allyl-2-(2-(diethylamino)ethoxy)-(DEAB)]]
*[[beta-xyloside]]
*[[blebbistatin]]
*[[BMS-453]]
*[[Brefeldin A (BFA)]]
*[[bumetanide]]
*[[calcium ionophore]]
*[[CGP54626 ]]
*[[Chelerythrine]]
*[[chenodeoxycholic acid]]
*[[chymostatin ]]
*[[Citral]]
*[[Colcemide]]
*[[concanamycin]]
*[[cycloheximide]]
*[[cyclopamine]]
*[[Cyclosporin A]]
*[[Cytochalasin B]]
*[[cytochalasin D]]
*[[D-AP5]]
*[[D-NAME]]
*[[Colcemide|Demecolcine]]
*[[deltamethrin]]
*[[dexamethosone]]
*[[DHT(dihydrotestosterone)]]
*[[diazepam]]
*[[Difopein]]
*[[Diphenyleneiodonium Chloride]]
*[[DMH-1]]
*[[Dorsomorphin]]
*[[DRB]]
*[[Ex-527]]
*[[finasteride]]
*[[folic acid]]
*[[forskolin]]
*[[fusicoccin]]
*[[gallein]]
*[[gentamycin]]
*[[GSK3-inhibitor IX (BIO)]]
*[[GM6001]]
*[[H-89 Dihydrochloride]]
*[[heptanol]]
*[[herbimycin A]]
*[[Hydroxyurea]]
*[[IBMX]]
*[[jasplakinolide]]
*[[6-B345TTQ]]
*[[KT 5720]]
*[[L-NAME]]
*[[lansoprazole]]
*[[latrunculin A]]
*[[latrunculin B]]
*[[LDN-193189]]
*[[leucovorin]]
*[[LiCl]]
*[[LY294002]]
*[[m-3M3FBS]]
*[[methotrexate (MTX)]]
*[[metronidazole]]
*[[Mg132]]
*[[MMP-2 inhibitor II]]
*[[MS-275]]
*[[nicorandil]]
*[[Nicotinamide]]
*[[Nicotinic acid]]
*[[nifedipine]]
*[[nocodazole]]
*[[NSC-87877]]
*[[o-phenanthroline]]
*[[okadaic acid]]
*[[olomoucine]]
*[[ouabain]]
*[[PD173074]]
*[[PD-407824]]
*[[p-MPPI]]
*[[taxol|Paclitaxel]]
*[[phaclofen]]
*[[Picropodophyllin]]
*[[PMA]]
*[[PP2]]
*[[primaquine]]
*[[Progesterone]]
*[[propylthiouracil]]
*[[pyrimethamine]]
*[[quinpirole]]
*[[rac1 inhibitor]]
*[[rapamycin]]
*[[retinoic acid]]
*[[Retinol]]
*[[RO-3306]]
*[[RS67333]]
*[[RU486]]
*[[SAHA]]
*[[SB 203580]]
*[[SB431542]]
*[[SB 505124]]
*[[SCH28080]]
*[[SNAP]]
*[[sodium nitroprusside]]
*[[SP600125 ]]
*[[SU5402]]
*[[SU6656]]
*[[Suramin]]
*[[tautomycin]]
*[[taxol]]
*[[thyroxine (T4)]]
*[[Torin 2]]
*[[Trichostatin A]]
*[[trimetrexate]]
*[[TTNPB]]
*[[Tubercidin]]
*[[tunicamycin]]
*[[Tyrphostin AG 1478]]
*[[U73122]]
*[[Y27632]]
*[[ZM 447439]]


IP Protocol
The Following are in need of standardization and curation
*[[AM251]]
This protocol utilizes the coupling of Antibody to Protein A Coated beads using DMP.  This allows for elution of bound proteins and reuse of beads, if desired. 
*[[AM281]]
*[[AP1510]]
*[[AR-A014418]]
*[[BHQ]]
*[[b-aminoproprionitrile]]
*[[beclomethasone]]
*[[Butyrate]]
*[[calcicludine]]
*[[Calyculin A]]
*[[celastrol]]
*[[celecoxib]]
*[[chromanol 293B]]
*[[chlorate]]
*[[cytochalasin D]]
*[[colchicine]]
*[[dimethyloxallyl glycine (DMOG)]]
*[[diclofenac]]
*[[etoposide]]
*[[IWR1]]
*[[GW843682X]]
*[[leptomycin B]]
*[[LY320135]]
*[[methylcellulose (MC)]]
*[[methimazole]]
*[[MBCD or MBetaCD]]
*[[MS222]]
*[[nicardipine]]
*[[Nu6102]]
*[[phenylthiourea|phenylthiourea (PTU)]]
*[[purmorphamine]]
*[[Pyrazoleanthrone]]
*[[R(+)BayK 8644]]
*[[RHC80267]]
*[[RL-3]]
*[[SAG]]
*[[SB-431542]]
*[[TG003]]
*[[Thapsigargin]]
*[[U0126]]
*[[ursodeoxycholic acid]]
*[[Echinomycin]]
*[[Gö 6983]]
*[[phenylhydrazine]]
*[[ω-conotoxin–GVIA]]
*[[flunarizine]]
*[[tetrodotoxin]]
*[[veratridine]]
*[[NBI-27914]]


Materials:
==Small Molecules Affecting Pathways==
===cAMP===
*[[IBMX]]
*[[RU486]]


Bio-Rad Protein A Support
===CDK/Cyclins===
*[[olomoucine]]
*[[RO-3306]]


Control IgG (depending on antibody source; rabbit, mouse, etc..)
===ERK/MAPK===
*[[U0126]]
===Folic Acid===
*[[Methotrexate (MTX)]]


PBSt (PBS + 0.1% Tween20)
===JAK/STAT===
*[[AZD1480]]


0.2 M Sodium Borate pH 9.0
===JNK===
*[[SP600125]]
*[[Pyrazoleanthrone]]


0.1M Glycine, pH 2.5
===Nitric Oxide===
*[[sodium nitroprusside]]


Trichloroacetic Acid (TCA)
===PDGF===
*[[AG1296]]
*[[AP1510]]


1% Deoxycholic acid (DOC)
===Retinoic Acid===
*[[AGN193109]]
*[[Citral]]
Methods
*[[retinoic acid]]
*[[Retinol]]
*[[TTNPB]]
*[[BMS-453]]


1. Remove an appropriate amount of Protein A beads and wash 3 times with PBSt. You will need a minimum of 2 tubes of beads at this point, one for you’re your antibody of interest and one for control IgG.
===Shh===
*[[cyclopamine]]
*[[purmorphamine]]
*[[Vismodegib]]


2. Assuming you are using 10-100 µl of beads, add PBSt and antibody to a final volume of ~500µl. 
===TGF-beta/BMP===
*[[SB-431542]]
*[[SB505124]]


3. Mix the antibody/bead solution in the cold room for 1 hour to overnight.
===Thyroid Signaling===
*[[PTU]]


4. Wash the beads 3 times with PBSt
===Wnt===
*[[LiCl]]
*[[GSK3-inhibitor IX (BIO)]]
*[[IWR1]]


5. Wash the beads 2 times with 0.2M Sodium Borate pH9.0
===Epigenetics===
*[[Ex-527]]


6. With beads in Borate, prepare 20mM solution of dimethylpimilidate (DMP) in sodium borate (you will need a minimum of 10 fold excess volume of this solution relative to your starting bead bed volume, for example 50ul beads require a minimum of 500 µl of 20mM DMP solution).
===Protease Inhibitors===
*[[chymostatin]]
*[[GM6001|GM6001 (MMP inhibitor)]]
*[[o-phenanthroline|o-phenanthroline(MMP inhibitor)]]
*[[BB-94|BB-94 MMP inhibitor]]
*[[GI254023X| GI254023X MMP10 inhibitor]]


7. Resuspend beads in at least a ten fold excess of Borate + 20mM DMP.  If I have less than 100µl of beads, I usually use 500-1000µl regardless.
==Small Molecules affecting Biological Functions & Processes==
===Angiogenesis===
*[[dimethyloxallyl glycine (DMOG)]]
*[[herbimycin A]]
*[[Suramin]]


8. Rotate at room temperature for 30 minutes.
===Apoptosis===
*[[cycloheximide]]
*[[alpha-amanitin]]
*[[Cyclosporin A]]
*[[2,2ʹ-Methylenebis(1,3-cyclohexanedione)]]
*[[NS3694]]


9. Spin down beads and wash 2-3 times in 100mM Tris pH8. This step quenches the coupling reaction. 
===Autophagy===
*[[LY-294002]]


10. Allow beads to rotate with tris for 2 hours at room temperature, or overnight in the cold room.
===Calcium Signaling===
*[[nifedipine]]
*[[R(+)BayK 8644]]
*[[nicardipine]]
*[[deltamethrin]]
*[[BHQ]]
*[[Thapsigargin]]


11. Wash beads 2-3 times in PBSt
===Circadian Rhythm===
*[[quinpirole]]


12. Wash beads 3 times in 0.1M glycine pH2.5.  This step removes any unbound antibody. 
===Cytoskeletal Inhibitors===
*[[Cytochalasin B]]
*[[cytochalasin D]]
*[[Colcemide]]
*[[jasplakinolide]] is an activator
*[[latrunculin B]]
*[[taxol]]
*[[taxol|Paclitaxel]]


13. Wash beads back into PBSt 3 times. The glycine will effectively denature the antibodies bound to beads, so it is important to let them refold in PBSt for a minimum of several hours to overnight in the cold.  If not the IP can be very dirty.
===Cell Cycle, including mitosis===
*[[Colcemide]]
*[[Hydroxyurea]]
*[[olomoucine]]
*[[PD-407824]]
===Cilia===
*[[methylcellulose (MC)]]


14. Wash beads in buffer of choice prior to IP.  Spin down and remove excess buffer. 
===DNA Damage/Repair===
*[[etoposide]]
*[[Hydroxyurea]]


15. Add solution you are IP-ing from to the beads, resuspend and incubate at either room temperature or in cold for a minimum of 1-2 hours.  The length of time for the IP depends on the quality of the antibody.  Also, things will get degraded faster (much faster) at room temperature.  I would recommend starting with 2-3 hours in the cold.
===DNA Synthesis===
*[[Calyculin A]]


16. After the IP, wash the beads 3 times in buffer (PBSt works, although you can wash with higher salt to be more stringent).
===Growth Cone, Filopodia, and other Cytoskeletal projections===
*[[PP2]]
*[[SU6656]]
*[[cytochalasin D]] 0.1 uM Sigma
*[[latrunculin B]] 30nM Sigma
*[[colchicine]] 12.5uM Sigma
*[[nocodazole]] 0.1uM Sigma
*[[RHC80267]]


17. Elute the beads 3 times in 0.1M Glycine pH 2.5.  Transfer the supernatant after each elution into 1.0M Tris pH8.  Normally, I will elute with 3 times, each with 200-300µl of glycine, and put all three elutions into 300µl of 1M Tris (final volume of 1.2ml).
===Hatching===
*[[ouabain]]


18. TCA precipitate samples by adding a 1:50 volume of 1% DOC and a 1:10 volume of TCA. Vortex quickly to mix. 
===Left-Right Patterning===
*[[methylcellulose (MC)]]


19. Store TCA precipitation on ice for 30 minutes to overnight (30 minutes is almost always sufficient). 
===Metamorphosis===
*[[3,3,5-triiodothyronine (T3)]]
*[[methimazole]]
*[[propylthiouracil]]
*[[thyroxine (T4)]]


20. Spin in a cold benchtop centrifuge at high speed for 15 minutes.
===Neurotransmission===
*[[diazepam]]
*[[p-MPPI]]
*[[phaclofen]]
*[[RS67333]]
*[[deltamethrin]]


21. Remove supernatant, add 500µl of acetome to pellet to remove salt and crap. 
===Protein Recycling===
*[[primaquine]]


22. Respin for 5-10 minutes on high speed.
==Drugs by Gene Class==


23. Remove all supernatant and resuspend in sample buffer. If sample buffer doesn’t stay blue, that is bad.
===Channels, including ATPases===
*[[ouabain]]
*[[AM251]]
*[[AM281]]
*[[LY320135]]
===G Protein Receptors===
*[[purmorphamine]]
===Kinases===
*[[KT 5720]]
*[[olomoucine]]
*[[PD-407824]]
*[[SB 203580]]
*[[Y27632]]
*[[ZM 447439]]
 
===Phosphatases===
*[[Cyclosporin A]]
*[[deltamethrin]]
*[[okadaic acid]]
*[[tautomycin]]
*[[Calyculin A]]
 
===Small GTPases===
 
 
===Protein Synthesis===
*[[anisomycin]] 40uM Sigma
*[[cycloheximide]] 40uM
 
===Retinoids===
*[[TTNPB]] Sigma
 
==Textpresso Small Molecules to Research==
*[http://www.xenbase.org/literature/article.do?method=display&articleId=43038 importazole inhibits importin]
*[http://www.xenbase.org/literature/article.do?method=display&articleId=42068 M119]
*[http://www.xenbase.org/literature/article.do?method=display&articleId=42068 GBG or GBetaGamma or G Protein Beta Gamma]
*[[LY-294002| LY-294002 inhibits PI3K and autophagy]]
 
==Models of Anemia==
*[[Phenylhydrazine]]

Revision as of 08:37, 7 November 2018

Notice to our users

Please help us to improve this small molecules wiki and our other wikis by registering for a wiki account through our normal feedback email.

Important small molecule references

Alphabetical List of Small Molecules With Literature References

The Following are in need of standardization and curation

Small Molecules Affecting Pathways

cAMP

CDK/Cyclins

ERK/MAPK

Folic Acid

JAK/STAT

JNK

Nitric Oxide

PDGF

Retinoic Acid

Shh

TGF-beta/BMP

Thyroid Signaling

Wnt

Epigenetics

Protease Inhibitors

Small Molecules affecting Biological Functions & Processes

Angiogenesis

Apoptosis

Autophagy

Calcium Signaling

Circadian Rhythm

Cytoskeletal Inhibitors

Cell Cycle, including mitosis

Cilia

DNA Damage/Repair

DNA Synthesis

Growth Cone, Filopodia, and other Cytoskeletal projections

Hatching

Left-Right Patterning

Metamorphosis

Neurotransmission

Protein Recycling

Drugs by Gene Class

Channels, including ATPases

G Protein Receptors

Kinases

Phosphatases

Small GTPases

Protein Synthesis

Retinoids

Textpresso Small Molecules to Research

Models of Anemia

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