Western blot protocol (Conlon lab) and Small Molecules for Xenopus Research: Difference between pages

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imported>Xenbase
 
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Protocol submitted by VGP from Conlon lab protocols [http://www.unc.edu/~fconlon/Protocols.htm#tropprot]
==Notice to our users==
Please help us to improve this small molecules wiki and our other wikis by registering for a wiki account through our normal feedback email.


==Important small molecule references==
*[http://www.xenbase.org/literature/article.do?method=display&articleId=35021 Database of Small Molecules by Feldman and Hogue 2006] at [https://pubchem.ncbi.nlm.nih.gov/ PubChem]
*[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]


link to protocol document [http://www.unc.edu/~fconlon/Protocols/Western%20and%20lysis.doc]
==Alphabetical List of Small Molecules With Literature References==


*[[1,3-Dicyclohexylurea]]
*[[11-cis retinal]]
*[[18alpha-glycyrretinic acid]]
*[[2'-deoxyadenosine]]
*[[2-APB]]
*[[3,3,5-triiodothyronine (T3)]]
*[[5HT (seratonin)]]
*[[8-Pcpt-2-O-Me-cAMP-AM]]
*[[activin]]
*[[AG1296]]
*[[AG1879]]
*[[AGN193109]]
*[[alpha-amanitin]]
*[[anandamide]]
*[[apelin]]
*[[aphidicolin]]
*[[AZD1480]]
*[[Bafilomycin A1]]
*[[BAPTA-AM]]
*[[Barasertib]]
*[[Benzaldehyde, 3-allyl-2-(2-(diethylamino)ethoxy)-(DEAB)]]
*[[beta-xyloside]]
*[[blebbistatin]]
*[[BMS-453]]
*[[Brefeldin A (BFA)]]
*[[bumetanide]]
*[[calcium ionophore]]
*[[CGP54626 ]]
*[[Chelerythrine]]
*[[chenodeoxycholic acid]]
*[[chymostatin ]]
*[[Citral]]
*[[Colcemide]]
*[[concanamycin]]
*[[cycloheximide]]
*[[cyclopamine]]
*[[Cyclosporin A]]
*[[Cytochalasin B]]
*[[cytochalasin D]]
*[[D-AP5]]
*[[D-NAME]]
*[[Colcemide|Demecolcine]]
*[[deltamethrin]]
*[[dexamethosone]]
*[[DHT(dihydrotestosterone)]]
*[[diazepam]]
*[[Difopein]]
*[[Diphenyleneiodonium Chloride]]
*[[DMH-1]]
*[[Dorsomorphin]]
*[[DRB]]
*[[Ex-527]]
*[[finasteride]]
*[[folic acid]]
*[[forskolin]]
*[[fusicoccin]]
*[[gallein]]
*[[gentamycin]]
*[[GSK3-inhibitor IX (BIO)]]
*[[GM6001]]
*[[H-89 Dihydrochloride]]
*[[heptanol]]
*[[herbimycin A]]
*[[Hydroxyurea]]
*[[IBMX]]
*[[jasplakinolide]]
*[[6-B345TTQ]]
*[[KT 5720]]
*[[L-NAME]]
*[[lansoprazole]]
*[[latrunculin A]]
*[[latrunculin B]]
*[[LDN-193189]]
*[[leucovorin]]
*[[LiCl]]
*[[LY294002]]
*[[m-3M3FBS]]
*[[methotrexate (MTX)]]
*[[metronidazole]]
*[[Mg132]]
*[[MMP-2 inhibitor II]]
*[[MS-275]]
*[[nicorandil]]
*[[Nicotinamide]]
*[[Nicotinic acid]]
*[[nifedipine]]
*[[nocodazole]]
*[[NSC-87877]]
*[[o-phenanthroline]]
*[[okadaic acid]]
*[[olomoucine]]
*[[ouabain]]
*[[PD173074]]
*[[PD-407824]]
*[[p-MPPI]]
*[[taxol|Paclitaxel]]
*[[phaclofen]]
*[[Picropodophyllin]]
*[[PMA]]
*[[PP2]]
*[[primaquine]]
*[[Progesterone]]
*[[propylthiouracil]]
*[[pyrimethamine]]
*[[quinpirole]]
*[[rac1 inhibitor]]
*[[rapamycin]]
*[[retinoic acid]]
*[[Retinol]]
*[[RO-3306]]
*[[RS67333]]
*[[RU486]]
*[[SAHA]]
*[[SB 203580]]
*[[SB431542]]
*[[SB 505124]]
*[[SCH28080]]
*[[SNAP]]
*[[sodium nitroprusside]]
*[[SP600125 ]]
*[[SU5402]]
*[[SU6656]]
*[[Suramin]]
*[[tautomycin]]
*[[taxol]]
*[[thyroxine (T4)]]
*[[Torin 2]]
*[[Trichostatin A]]
*[[trimetrexate]]
*[[TTNPB]]
*[[Tubercidin]]
*[[tunicamycin]]
*[[Tyrphostin AG 1478]]
*[[U73122]]
*[[Y27632]]
*[[ZM 447439]]


Western Protocol
The Following are in need of standardization and curation
*[[AM251]]
*[[AM281]]
*[[AP1510]]
*[[AR-A014418]]
*[[BHQ]]
*[[b-aminoproprionitrile]]
*[[beclomethasone]]
*[[Butyrate]]
*[[calcicludine]]
*[[Calyculin A]]
*[[celastrol]]
*[[celecoxib]]
*[[chromanol 293B]]
*[[chlorate]]
*[[cytochalasin D]]
*[[colchicine]]
*[[dimethyloxallyl glycine (DMOG)]]
*[[diclofenac]]
*[[etoposide]]
*[[IWR1]]
*[[GW843682X]]
*[[leptomycin B]]
*[[LY320135]]
*[[methylcellulose (MC)]]
*[[methimazole]]
*[[MBCD or MBetaCD]]
*[[MS222]]
*[[nicardipine]]
*[[Nu6102]]
*[[phenylthiourea|phenylthiourea (PTU)]]
*[[purmorphamine]]
*[[Pyrazoleanthrone]]
*[[R(+)BayK 8644]]
*[[RHC80267]]
*[[RL-3]]
*[[SAG]]
*[[SB-431542]]
*[[TG003]]
*[[Thapsigargin]]
*[[U0126]]
*[[ursodeoxycholic acid]]
*[[Echinomycin]]
*[[Gö 6983]]
*[[phenylhydrazine]]
*[[ω-conotoxin–GVIA]]
*[[flunarizine]]
*[[tetrodotoxin]]
*[[veratridine]]
*[[NBI-27914]]


(updated 3-17-2011)
==Small Molecules Affecting Pathways==
===cAMP===
*[[IBMX]]
*[[RU486]]


===CDK/Cyclins===
*[[olomoucine]]
*[[RO-3306]]


FOR EMBRYOS:
===ERK/MAPK===
*[[U0126]]
===Folic Acid===
*[[Methotrexate (MTX)]]


Lysis Buffer (10 ml)
===JAK/STAT===
*[[AZD1480]]


{|border="1" cellpadding="5"
===JNK===
|-
*[[SP600125]]
! scope="row" style="background:#efefef;" |Lysis Buffer (10 ml)
*[[Pyrazoleanthrone]]
|500ul 1M Tris pH 7.6
|-
! scope="row" style="background:#efefef;" |150mM NaCl
|200ul 0.5M EDTA
|-
! scope="row" style="background:#efefef;" |10mM EDTA
|200ul 0.5M EDTA
|-
! scope="row" style="background:#efefef;" |1% TritonX-100
|1 ml 10% TritonX-100
|-
! scope="row" style="background:#efefef;" |Protease inhibitors
|1 Roche complete mini tablet (4°)
|}


===Nitric Oxide===
*[[sodium nitroprusside]]


===PDGF===
*[[AG1296]]
*[[AP1510]]


Lysis and SDS PAGE
===Retinoic Acid===
*[[AGN193109]]
*[[Citral]]
*[[retinoic acid]]
*[[Retinol]]
*[[TTNPB]]
*[[BMS-453]]


1. Turn on Lieb lab water bath biorupter about 30min before lysis.
===Shh===
*[[cyclopamine]]
*[[purmorphamine]]
*[[Vismodegib]]


2. Lyse embryos on ice in 100ul of cold lysis buffer by pipetting up and down.
===TGF-beta/BMP===
*[[SB-431542]]
*[[SB505124]]


3. Sonicate samples for 10min on High setting: 30sec on/ 30sec off.
===Thyroid Signaling===
*[[PTU]]


4. Spin samples at 4° at 14,000 rpm for 5min.
===Wnt===
*[[LiCl]]
*[[GSK3-inhibitor IX (BIO)]]
*[[IWR1]]


5. Move supernatant to new tube. 
===Epigenetics===
*[[Ex-527]]


6. Normalize loading volumes by performing a Coomassie protein assay for all samples.
===Protease Inhibitors===
*[[chymostatin]]
*[[GM6001|GM6001 (MMP inhibitor)]]
*[[o-phenanthroline|o-phenanthroline(MMP inhibitor)]]
*[[BB-94|BB-94 MMP inhibitor]]
*[[GI254023X| GI254023X MMP10 inhibitor]]


7. For maximum loading volume combine 17.75ul protein (17.75ul of sample with lowest concentration, then normalize other samples to this using water to make up the difference) with 6.25ul 4X Loading Dye and 1ul BME. 
==Small Molecules affecting Biological Functions & Processes==
===Angiogenesis===
*[[dimethyloxallyl glycine (DMOG)]]
*[[herbimycin A]]
*[[Suramin]]


8. Boil samples for 6-10 minutes.
===Apoptosis===
*[[cycloheximide]]
*[[alpha-amanitin]]
*[[Cyclosporin A]]
*[[2,2ʹ-Methylenebis(1,3-cyclohexanedione)]]
*[[NS3694]]


9. Spin samples at 14,000 rpm, RT for 3 min.
===Autophagy===
*[[LY-294002]]


10. Load 25ul of sample onto gel for western analysis.
===Calcium Signaling===
*[[nifedipine]]
*[[R(+)BayK 8644]]
*[[nicardipine]]
*[[deltamethrin]]
*[[BHQ]]
*[[Thapsigargin]]


===Circadian Rhythm===
*[[quinpirole]]


FOR TISSUE CULTURE:
===Cytoskeletal Inhibitors===
*[[Cytochalasin B]]
*[[cytochalasin D]]
*[[Colcemide]]
*[[jasplakinolide]] is an activator
*[[latrunculin B]]
*[[taxol]]
*[[taxol|Paclitaxel]]


:RIPA Buffer (100 mL – can store at 4°)
===Cell Cycle, including mitosis===
*[[Colcemide]]
*[[Hydroxyurea]]
*[[olomoucine]]
*[[PD-407824]]
===Cilia===
*[[methylcellulose (MC)]]


:Add 790mg Tris base to 75mL diH20.  (50mM Tris-HCl, pH 7.4)
===DNA Damage/Repair===
*[[etoposide]]
*[[Hydroxyurea]]


:Add 900mg NaCl and stir until all solids are dissolved. (150mM NaCl)
===DNA Synthesis===
*[[Calyculin A]]


:Using HCl, adjust pH to 7.4
===Growth Cone, Filopodia, and other Cytoskeletal projections===
*[[PP2]]
*[[SU6656]]
*[[cytochalasin D]] 0.1 uM Sigma
*[[latrunculin B]] 30nM Sigma
*[[colchicine]] 12.5uM Sigma
*[[nocodazole]] 0.1uM Sigma
*[[RHC80267]]


:Add 10mL 10% NP-40 (1% NP-40)
===Hatching===
*[[ouabain]]


:Add 2.5mL 10% Na-deoxycholate and stir until solution is clear (0.25% Na-deoxych.)
===Left-Right Patterning===
*[[methylcellulose (MC)]]


:Add 1mL 100mL EDTA.  (1mM EDTA)
===Metamorphosis===
*[[3,3,5-triiodothyronine (T3)]]
*[[methimazole]]
*[[propylthiouracil]]
*[[thyroxine (T4)]]


:Bring to 100mL with diH20.
===Neurotransmission===
*[[diazepam]]
*[[p-MPPI]]
*[[phaclofen]]
*[[RS67333]]
*[[deltamethrin]]


===Protein Recycling===
*[[primaquine]]


At time of use, add protease inhibitors
==Drugs by Gene Class==


Lysis and SDS PAGE
===Channels, including ATPases===
*[[ouabain]]
*[[AM251]]
*[[AM281]]
*[[LY320135]]
===G Protein Receptors===
*[[purmorphamine]]
===Kinases===
*[[KT 5720]]
*[[olomoucine]]
*[[PD-407824]]
*[[SB 203580]]
*[[Y27632]]
*[[ZM 447439]]


1. Aspirate media from cells. Wash cells with ice cold PBS 1X.
===Phosphatases===
*[[Cyclosporin A]]
*[[deltamethrin]]
*[[okadaic acid]]
*[[tautomycin]]
*[[Calyculin A]]


2. Add 200uL RIPA/well of 6-well dish (just enough RIPA to barely cover cells)
===Small GTPases===


3. Rock plate at 4° for 5min.


4. Rinse cells off plate using RIPA buffer. Alternately, scrape cells from well in PBS, then spin at 1500 rpm for 10min at 4°, aspirate PBS from pellet, and resuspend pellet in desired amount of RIPA.
===Protein Synthesis===
*[[anisomycin]] 40uM Sigma
*[[cycloheximide]] 40uM


5. Transfer lysate to eppendorf. Sonicate samples for 5min (30 sec on/30 sec off) on High in Lieb lab biorupter.
===Retinoids===
*[[TTNPB]] Sigma


6. Spin samples at 14,000 rpm for 5min at 4°.
==Textpresso Small Molecules to Research==
*[http://www.xenbase.org/literature/article.do?method=display&articleId=43038 importazole inhibits importin]
*[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]]


7. Move supernatant to new tube.
==Models of Anemia==
 
*[[Phenylhydrazine]]
8. Normalize loading volumes by performing a Coomassie protein assay for all samples.
 
9. Combine desired amount of protein (minimum of 10ug) up to 17.75ul (normalize all samples so that each samples contains the same amount of total protein) with 6.25ul 4X Loading Dye and 1ul BME.
 
10. Boil samples 6-10 minutes.
 
11. Spin samples at 14,000rpm for 3min at RT.
 
12. Load 25ul onto gel.
 
 
Transfer
 
1.  Build “gel sandwich” in transfer buffer using two pieces of Whatman paper, a nitrocellulose membrane, and two sponges.  I find it easiest to build using this orientation:  Gray side-sponge-Whatman paper-gel-nitrocellulose-Whatman paper-sponge-clear side.  Gray side faces black electrode! Don’t forget to orient the gel on the membrane so that the lanes do not transfer backwards. 
 
 
Transfer Buffer
 
:15g glycine
 
:3.5g Tris base
 
:Bring to 800ml with diH20
 
:Add 200ml MeOH
 
 
2.  Transfer gel ON at 30V or for 1 hr at 400 mA at 4°. 
 
3.  Transfer membrane to TBST for storage or to blocking solution.
 
 
Immunoblotting
 
1. Block membrane in 5% dry milk in TBST for at least 1 hour at RT or ON at 4°
 
2. Wash membrane 3X in TBST, 5-15min/wash
 
3. Apply primary antibody diluted in Blocking Solution ON at 4° or for 2hrs at RT.
 
4. Wash membrane 3X in TBST, 5-15min/wash
 
5. Apply secondary antibody diluted in 5% milk in TBST for 1h at RT.
 
6. Wash membrane 4X in TBST, 5-15min/wash.
 
7. Develop with ECL.

Revision as of 08:37, 7 November 2018

Notice to our users

Please help us to improve this small molecules wiki and our other wikis by registering for a wiki account through our normal feedback email.

Important small molecule references

Alphabetical List of Small Molecules With Literature References

The Following are in need of standardization and curation

Small Molecules Affecting Pathways

cAMP

CDK/Cyclins

ERK/MAPK

Folic Acid

JAK/STAT

JNK

Nitric Oxide

PDGF

Retinoic Acid

Shh

TGF-beta/BMP

Thyroid Signaling

Wnt

Epigenetics

Protease Inhibitors

Small Molecules affecting Biological Functions & Processes

Angiogenesis

Apoptosis

Autophagy

Calcium Signaling

Circadian Rhythm

Cytoskeletal Inhibitors

Cell Cycle, including mitosis

Cilia

DNA Damage/Repair

DNA Synthesis

Growth Cone, Filopodia, and other Cytoskeletal projections

Hatching

Left-Right Patterning

Metamorphosis

Neurotransmission

Protein Recycling

Drugs by Gene Class

Channels, including ATPases

G Protein Receptors

Kinases

Phosphatases

Small GTPases

Protein Synthesis

Retinoids

Textpresso Small Molecules to Research

Models of Anemia

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