Isolation of mitotic chromosomes from XTC cells (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]]


Purification of Mitotic Chromosomes-
===PDGF===
Mitchison and Desai Method
*[[AG1296]]
===Retinoic Acid===
*[[AGN193109]]
DAY BEFORE
*[[retinoic acid]]
*[[TTNPB]]
===Shh===
*[[cyclopamine]]
*[[mHip]]
===TGF-beta/BMP===
===Wnt===
*[[LiCl]]


1. Arrest XTC cells (10-15 plates) with a final concentration of 1 µg/ml of Nocodazole (1µl per 10 ml of culture media) overnight (~16hrs).
===Cytoskeletal Inhibitors===
*[[Cytochalasin B]]
2. Make up 10x Swelling Buffer (1x = 5 mM PIPES, 5 mM NaCl, 5 mM MgCl2, 1 mM EGTA,  pH 7.2).  Dilute a total of 150 ml to 1x, keeping most at RT and about 25 ml on ice. 
===Epigenetics===
[[Ex-527]]
3. Chill HB-4 in centrifuge to 4°C.  Put 2 x 15 ml corex tubes on ice.  Chill 7 ml dounce homogenizer and tight fitting pestle on ice.  Thaw Hoechst. Make up solutions for sucrose gradient steps (see Buffers at end).  Turn on microscope in Burke Lab. 
===Protease Inhibitors===
*[[chymostatin]]
4. Collect mitotic cells by mitotic shake / blow off.
*[[GM6001|GM6001 (MMP inhibitor)]]
*[[o-phenanthroline|o-phenanthroline(MMP inhibitor)]]
5. Pellet cells by using Jouan CR4.12 centrifuge in 50ml tubes at ~2,000 rpm for 3 minutes.
6. Resuspend in 10 ml of RT  Swelling buffer.
7. After the pellet is resuspended, add 40 ml more of RT swelling buffer.
8. Swell at RT for ~5 minutes. 
9. Prepare lysis buffer described below and make up sucrose step gradient during swelling.
10. Pellet (same as above) and resuspend vigorously in 7 ml of ice cold lysis buffer (5 mM PIPES, 5 mM NaCl, 5 mM MgCl2, 1 mM EGTA,  pH 7.2 plus 0.1% Digitonin and LPC).  Excess digitonin is spun out (or settles out before use). 
11. Transfer the cells to a chilled 7 ml dounce homogenizer and disrupt with 15-20 strokes using a tight pestle, resting for 2 seconds after each dounce.
12. Add Hoeschst to 2µg/ml and check by fluorescence to see that spindles have been disrupted. 
13. Transfer to a 14ml round bottom, polypro falcon tube (#352029) and spin in an HB-4 at ~900 rpm for 1 min at 4°C to pellet interphase nuclei and debris.
14. Layer the supernatant (~6 ml) onto 30/40/50/60% (2ml, 2ml, 2ml, 3ml) sucrose step gradient (made up with in swelling buffer) in a chilled 15 ml corex tube. 
15. Centrifuge at 5k for 15 minutes and 4°C in HB-4 swinging bucket rotor, brake off.
16. Collect chromosome in flocculent white mass using a pasteur pipette from the interphase between layers of sucrose. 
17. Check chromosomes again with Hoechst.  If there are a lot of interphase nuclei still present spin gently (~500 x g, 30 seconds) in a chilled benchtop centrifuge.
18. Freeze mitotic chromosome in lN2.
Buffers
Swelling Buffer (1x):
5 mM PIPES, pH 7.2
5 mM NaCl
5 mM MgCl2
1 mM EGTA
Lysis Buffer:
5 mM PIPES, pH 7.2
5 mM NaCl
5 mM MgCl2
1 mM EGTA


LPC .1% Digitonin
 
===Small Molecules Studied in Oocytes Found Via Textpresso===
Stock Hoechst- 10 mg/ml
*[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]
Stock Nocodazole- 10mg/ml
*[[LY-294002| LY-294002 inhibits PI3K and autophagy]]
 
Coffee- Medium Roast + Half and Half + Sugar
===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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