Cycling extracts, sara's protocol (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]
==Important small molecule references==
*[http://www.xenbase.org/literature/article.do?method=display&articleId=35021 Database of Small Molecules by Feldman and Hogue 2006]
*[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]


==Alphabetical List of Small Molecules With Literature References==


link to protocol page [http://people.virginia.edu/~djb6t/LabWeb/frogs.htm]
*[[11-cis retinal]]
*[[18alpha-glycyrretinic acid]]
*[[3,3,5-triiodothyronine (T3)]]
*[[5HT (seratonin)]]
*[[activin]]
*[[AG1296]]
*[[AG1879]]
*[[AGN193109]]
*[[alpha-amanitin]]
*[[anandamide]]
*[[apelin]]
*[[aphidicolin ]]
*[[AZD1480]]
*[[BAPTA-AM]]
*[[beta-xyloside]]
*[[bumetanide]]
*[[calcium ionophore]]
*[[CGP54626 ]]
*[[Chelerythrine]]
*[[chymostatin ]]
*[[concanamycin]]
*[[cycloheximide]]
*[[cyclopamine]]
*[[Cytochalasin B]]
*[[dexamethosone]]
*[[DHT(dihydrotestosterone)]]
*[[Ex-527]]
*[[forskolin]]
*[[fusicoccin]]
*[[gentamycin]]
*[[GM6001]]
*[[heptanol]]
*[[IBMX]]
*[[KT 5720]]
*[[lansoprazol]]
*[[LiCl]]
*[[Mg132]]
*[[mHip]]
*[[MMP-2 inhibitor II]]
*[[nicorandil]]
*[[nifedipine]]
*[[nocodazole]]
*[[NSC-87877]]
*[[o-phenanthroline]]
*[[olomoucine]]
*[[ouabain]]
*[[p-MPPI]]
*[[phaclofen]]
*[[PMA]]
*[[PP2]]
*[[primaquin]]
*[[propythiouracil]]
*[[retinoic acid]]
*[[RS67333]]
*[[RU486]]
*[[SB 203580]]
*[[sodium nitroprusside]]
*[[SP600125 ]]
*[[SU5402]]
*[[SU66056]]
*[[Suramin]]
*[[thyroxine (T4)]]
*[[TTNPB]]
*[[tunicamycin]]
*[[Y27632]]
*[[ZM 447439]]


==List of Molecules Affecting Specific Pathways==
Sara's Cycling Xenopus Extracts
===cAMP===
[[IBMX]]
===ERK/MAPK===
===JAK/STAT===
*[[AZD1480]]
===JNK===
[[SP600125]]


To prepare: 5 days before the experiment, prime female frogs with 50 IU of PMSG.
===PDGF===
*[[AG1296]]
The night before the experiment, induce ovulation by injecting the primed frogs with 500 IU of HCG.  Place each frog in 2L of 1X MMR overnight at 18°C.
===Retinoic Acid===
*[[AGN193109]]
In the morning, collect laid eggs.  Place the frogs back into 1X MMR and continue to collect eggs. 
*[[retinoic acid]]
*[[TTNPB]]
Once you have sufficient amounts of eggs, wash the eggs in 1X MMR to remove debris and bad eggs.
===Shh===
*[[cyclopamine]]
Remove as much MMR as possible and de-jelly the eggs with 2% cysteine solution prepared in 1X XB salts.  Swirl eggs occasionally and de-jelly until the eggs pack tightly.  Pour off de-jelly, and rinse briefly with fresh de-jelly solution. 
*[[mHip]]
===TGF-beta/BMP===
===Wnt===
*[[LiCl]]


Wash the eggs into 1X MMR.  Activate the eggs by addition of 2μg/mL A23187, a calcium ionophore. (To 10 mL MMR, add 1μL ionophore).  Watch for activation (takes about 10 minutes).  Upon activation, wash out ionophore liberally with MMR.  Place the eggs at 18°C for 50 minutes.
===Cytoskeletal Inhibitors===
*[[Cytochalasin B]]
Wash the eggs 4X with XB, and 2X with XB+protease inhibitors (10μg/mL each leupeptin, pepstatin and chymostatin (LPC)). Load the eggs gently into ultraclear centrifuge tubes containing 1 mL XB+PIs and 100μg/mL cytochalasin B or D.
===Epigenetics===
[[Ex-527]]
Pack the eggs by spinning at low speed in a clinical centrifuge for 30-60 seconds. Aspirate off the buffer and spin at moderate speed in the clinical centrifuge for 30-60 seconds. Aspirate off residual buffer.
===Protease Inhibitors===
*[[chymostatin]]
Crush eggs by spinning in the mini ultracentrifuge swinging bucket rotor (RP55S-344) at 10,000 rpm for 10 minutes at 2°C.
*[[GM6001|GM6001 (MMP inhibitor)]]
*[[o-phenanthroline|o-phenanthroline(MMP inhibitor)]]
Remove the cytoplasmic fraction with a 1mL syringe and a 18-guage needle. 
 
 
Add 1/1000 volume of LPC, 1/1000 volume of cytochalasin B or D, and 1/40 energy mix and 1/40 sucrose to the cytoplasmic fraction.  Invert and flick gently to mix.
===Small Molecules Studied in Oocytes Found Via Textpresso===
*[http://www.xenbase.org/literature/article.do?method=display&articleId=42068 M119]
The extract is now ready for use.
*[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]]
 
===Textpresso Small Molecules to Research===
*[http://www.xenbase.org/literature/article.do?method=display&articleId=43038 importazole inhibits importin]

Revision as of 11:08, 2 June 2015

Important small molecule references

Alphabetical List of Small Molecules With Literature References

List of Molecules Affecting Specific Pathways

cAMP

IBMX

ERK/MAPK

JAK/STAT

JNK

SP600125

PDGF

Retinoic Acid

Shh

TGF-beta/BMP

Wnt

Cytoskeletal Inhibitors

Epigenetics

Ex-527

Protease Inhibitors


Small Molecules Studied in Oocytes Found Via Textpresso

Textpresso Small Molecules to Research

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