Silver staining protein gels (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==
===cAMP===
[[IBMX]]
===ERK/MAPK===
===JAK/STAT===
*[[AZD1480]]
===JNK===
[[SP600125]]


Silver Staining Protocol (Not Compatible with Mass Spec)
===PDGF===
*[[AG1296]]
===Retinoic Acid===
*[[AGN193109]]
*[[retinoic acid]]
*[[TTNPB]]
===Shh===
*[[cyclopamine]]
*[[mHip]]
===TGF-beta/BMP===
===Wnt===
*[[LiCl]]


===Cytoskeletal Inhibitors===
*[[Cytochalasin B]]
===Epigenetics===
[[Ex-527]]
===Protease Inhibitors===
*[[chymostatin]]
*[[GM6001|GM6001 (MMP inhibitor)]]
*[[o-phenanthroline|o-phenanthroline(MMP inhibitor)]]


Notes:
Be careful not to touch gel during any step of the procedure with either a gloved or ungloved hand, finger etc…
Use MilliQ-H2O for all steps
Perform all incubations in well washed glass dishes.


===Small Molecules Studied in Oocytes Found Via Textpresso===
*[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]]


1. Remove gels from glass plates in which they were run:
===Textpresso Small Molecules to Research===
*[http://www.xenbase.org/literature/article.do?method=display&articleId=43038 importazole inhibits importin]
2. Incubate in 50% Methanol, 10% Glacial Acetic Acid, 40% H2O for 30 minutes (100 ml)
3. Incubate in 5% Methanol, 7% Acetic Acid (Glacial), 88% H2O for 30 minutes (100ml)
4. Incubate in 10% Gluteraldehyde made up in H2O from 25% stock (25 ml) for 30 minutes.
5. Wash 4 x 20-30 minutes in H2O using 200-300 ml per wash.
6. Makes 2 Solutions, A and B:    A) 3 ml of 30% Ammonia Hydroxide, 0.25 g NaOH in 190 ml H2O.  B) 1.2g Silver Nitrate (AgNO3) into 10 ml of H2O. 
7. Add solution B to A slowly, mix gently, and add to gel, incubate for 30 minutes.
8. Make developer and stop solutions (see below).
9. Perform 5 x 1 minute washes with H2O, using 200 ml’s per wash.
10. Add developer solution (~100 ml) and wait and watch gel until you see bands come up.  Quickly remove developer solution and add stop solution.  (Developer Solution: 0.1g Citric Acid, 1ml 37% formaldehyde into 1L H2O. Stop Solution: 190 ml H2O and 10 ml Acetic Acid, Glacial)
11. After 10 minutes in Stop Solution wash thoroughly

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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