Cycling extracts, mike's protocol (Stukenberg lab) and Small Molecules for Xenopus Research: Difference between pages

From XenWiki
(Difference between pages)
Jump to navigation Jump to search
imported>Virgilio
 
imported>Kevin
No edit summary
 
Line 1: Line 1:
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]]


===PDGF===
Mike’s Cycling Xenopus Extracts
*[[AG1296]]
===Retinoic Acid===
*[[AGN193109]]
1. Prepare extracts as is done normally.  (Murray, 1991;Desai et al., 1999;Chen and Murray, 1997)  Do not centrifuge faster than 10K for 10 min at 16 deg C in HB-4 rotor (or equivalent in mini-ultra).  Higher speed spins will not cycle as well (or so I have read). 
*[[retinoic acid]]
*[[TTNPB]]
2. Supplement the extract with cytochalasin D, 1 X energy mix, 50 mM Sucrose and LPC.  The use of cytochalasin seems to help extracts cycle.  Although, since it is more expensive, I would use it for cycling extracts only, and not for normal CSF.
===Shh===
*[[cyclopamine]]
3. Bring aliquots of extract to room temperature for 1-2 minutes.  Add sperm at a concentration of  less than 1,000 sperm/ul (this can be varied, but I have read to high can overwhelm the extract) and gently flick the tube to thoroughly mix them the sperm in the extract.  Good mixing is essential, but the extract is sensitive, so be careful. 
*[[mHip]]
===TGF-beta/BMP===
4. Add 1/10 volume of cycling solution which contains 4 mM CaCl2, 100 mM KCl, 1 mM MgCl2. Again gently flick the tube to mix thoroughly. 
===Wnt===
*[[LiCl]]
5. At about 30-45 minutes the extracts will have exited morphologically.  This can be checked by squashing 1 µl under 3 µl of squash fix (MMR + ~10% formaldehyde + ~50% Glycerol + Hoechst).  The chromatin will appear rounded and diffuse and they should be uniform throughout the extract.  That is, all nuclei should be at almost the same point. 
 
===Cytoskeletal Inhibitors===
6. Check the extract every 10-15 minutes on the microscope. 
*[[Cytochalasin B]]
===Epigenetics===
7. When the chromatin is again condensed (around 90-120 minutes) add back an equal volume of room temperature CSF extract to re-arrest the cycling extract in mitosis.  After -15-20 minutes check to be sure that the chromatin is condensed in a in a tight mass. 
[[Ex-527]]
===Protease Inhibitors===
8. Add nocodazole (if required for experiment) to a final concentration of 10 µg/ml (dilute nocodazole in extract).
*[[chymostatin]]
 
*[[GM6001|GM6001 (MMP inhibitor)]]
9. Continue to incubate at room temperature for 15 minutes.
*[[o-phenanthroline|o-phenanthroline(MMP inhibitor)]]
 
References
 
===Small Molecules Studied in Oocytes Found Via Textpresso===
      1.   Chen, R.H. and A.Murray. 1997. Characterization of spindle assembly checkpoint in Xenopus egg extracts. Methods Enzymol. 283:572-584.
*[http://www.xenbase.org/literature/article.do?method=display&articleId=42068 M119]
      2.    Desai, A., A.Murray, T.J.Mitchison, and C.E.Walczak. 1999. The use of Xenopus egg extracts to study mitotic spindle assembly and function in vitro. Methods Cell Biol. 61:385-412.
*[http://www.xenbase.org/literature/article.do?method=display&articleId=42068 GBG or GBetaGamma or G Protein Beta Gamma]
      3.   Murray, A.W. 1991. Cell cycle extracts. Methods Cell Biol. 36:581-605.
*[[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

Retrieved from "https://wiki.xenbase.org/xenwiki/index.php?title=Cycling_extracts,_mike%27s_protocol_(Stukenberg_lab)&oldid=47515"