CSF extract, sara's protocol (Stukenberg lab) and Small Molecules for Xenopus Research: Difference between pages

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imported>Virgilio
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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]]


===PDGF===
*[[AG1296]]
===Retinoic Acid===
*[[AGN193109]]
*[[retinoic acid]]
*[[TTNPB]]
===Shh===
*[[cyclopamine]]
*[[mHip]]
===TGF-beta/BMP===
===Wnt===
*[[LiCl]]


Sara's CSF EXTRACT
===Cytoskeletal Inhibitors===
*[[Cytochalasin B]]
Preparation:
===Epigenetics===
[[Ex-527]]
2-3 L of 1X MMR, make night before, store at 20°C.
===Protease Inhibitors===
*[[chymostatin]]
*[[GM6001|GM6001 (MMP inhibitor)]]
*[[o-phenanthroline|o-phenanthroline(MMP inhibitor)]]


200mL de-jelly solution, 2% cysteine, pH to 7.8 with 0.9mL 10N NaOH.


750mL XB:
===Small Molecules Studied in Oocytes Found Via Textpresso===
10mM K-HEPES
*[http://www.xenbase.org/literature/article.do?method=display&articleId=42068 M119]
50mM sucrose
*[http://www.xenbase.org/literature/article.do?method=display&articleId=42068 GBG or GBetaGamma or G Protein Beta Gamma]
1X XB
*[[LY-294002| LY-294002 inhibits PI3K and autophagy]]
11uL 10N KOH per 100mL to keep pH 7.7


250mL CSF-XB:
===Textpresso Small Molecules to Research===
250mL XB (from above)
*[http://www.xenbase.org/literature/article.do?method=display&articleId=43038 importazole inhibits importin]
5mM EGTA
1mM MgCl2
 
100mL CSF-XB + PIs:
100mL CSF-XB (from above)
10 ug/mL LPC
 
2M Sucrose
 
LPC
 
Cytochalasin
 
40X Energy Mix
 
Sperm
 
Extract Fix:
60% Glycerol
1X MMR
1ug/mL Hoescht
10% Formaldehyde
 
Sperm Dilution Buffer (5X)
 
10X Calcium: 4mM CaCl2 in 1X Sperm Dilution Buffer
If the extracts will be spun onto coverslips, then also need:
 
Fixation Buffer:
BRB80
20% Glycerol
0.5% TX-100
4% Formaldehyde
 
Cushion:
BRB80
30% Glycerol
 
-20°C MeOH
 
 
Making a CSF Extract
Collect eggs.
 
Wash eggs in MMR 2-3X.
 
Remove as much MMR as possible, and pour on dejellying solution.
 
De-jelly 7-10 min.  Rinse 1X in de-jelly. While eggs are de-jellying, add 1mL CSF-XB +PIs to centrifuge tubes.  Add 10uL 10mg/mL cytochalasin and flick tube well.
 
Wash de-jellied eggs 3-4X in XB. 
 
Wash eggs 2-3 X in CSF-XB.
 
Wash eggs 2X in CSF-XB + PIs.  Leave in small volume of CSF-XB+PIs.
 
Gently pipet eggs into centrifuge tubes, put pipet below meniscus.
 
Remove excess buffer.
 
Spin 10 sec at 1500 rpm (setting 4) in clinical centrifuge/
 
Aspirate off buffer. Add 100uL Versilube to top
 
Pack eggs: spin at 2000rpm (setting 5) for 30 sec and full speed for 1min in clinical centrifuge.
 
Aspirate off versilube and buffer from top of eggs.
 
Centrifuge packed eggs at 10,000 rpm for 15 min at 16°C in the SW55.1 rotor.
 
Collect cytoplasmic layer of extract with 18-guage needle into a 1-cc syringe.  Avoid bubbles. 
 
Transfer to new tube and estimate volume.
 
Add 1/1000 volume LPC.
 
Add 1/1000 volume cytochalasin.
 
1/40 volume 40X energy mix.
 
Add 1/40 volume 2M sucrose.

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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