Immunofluorescence with biotinylated antibodies (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]
==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 page [http://people.virginia.edu/~djb6t/LabWeb/frogs.htm]
==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]]


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]]


Immunofluorescence with Biotinylated Antibodies
==Small Molecules Affecting Pathways==
===cAMP===
*[[IBMX]]
*[[RU486]]


===CDK/Cyclins===
1. Fix cells onto coverslips as normal, and wash appropriately.
*[[olomoucine]]
*[[RO-3306]]
2. Add to your normal first block (20% serum, or 3%BSA, etc., in your buffer of interest; I use PBST or TBST depending on the antibody), approximately 10ul/100ul of Avidin blocking solution (Vector Laboratories, catalog # SP-2001)
 
Block as usual, ~30 minutes at room temperature.
===ERK/MAPK===
*[[U0126]]
3. Incubate your coverslips in your 1st rabbit primary antibody as usual, (5% serum in your chosen buffer, etc.) at room temperature for 1hour.
===Folic Acid===
*[[Methotrexate (MTX)]]
4. Wash the coverslips twice for 5 minutes each in PBST or your buffer of choice.  I rock my coverslips gently in 6 or 12 well dishes on a rotator.
 
===JAK/STAT===
5. Add 10uL/100uL of Biotin blocking solution to the 1st anti-rabbit 2? antibody, and label the coverslips as usual (5% serum in your chosen buffer), ~45 minutes at room temperature in the dark to protect the fluorophore.
*[[AZD1480]]
 
6. Wash coverslips twice for 5 minutes each in PBST or your chosen buffer.  Keep the coverslips covered (I use some foil) to protect your fluorophore.
===JNK===
*[[SP600125]]
7. This is the critical step: Block the coverslips in rabbit serum or IgG.  This will block any leftover IgG from the first anti-rabbit secondary antibody that might be recognized by the biotinylated rabbit antibody that you will use in the next step.  If you forget this step, your results will be skewed significantly by the high background staining resulting from the biotinylated rabbit antibody binding to your anti-rabbit secondary used in the previous step.
*[[Pyrazoleanthrone]]
 
8. Probe the coverslips with your biotinylated 2nd primary rabbit antibody (5% serum in buffer), 1 hour at room temperature in the dark.
===Nitric Oxide===
*[[sodium nitroprusside]]
9. Wash coverslips twice for 5 minutes each in PBST or your chosen buffer.  Keep the coverslips covered to protect your fluorophore.
 
===PDGF===
10. Incubate the coverslips with Avidin-Cy3 or Avidin-FITC as your 2nd rabbit antibody secondary, for ~45 minutes at room temperature in the dark.  (I generally use Avidin-Cy3 (Sigma E-4142) at 1:5,000).
*[[AG1296]]
*[[AP1510]]
11. Wash coverslips as before, then wash coverslips 1X with ddH2O.
 
===Retinoic Acid===
13. Stain coverslips with DAPI or Hoescht 33342.
*[[AGN193109]]
*[[Citral]]
14. Mount coverslips with mounting media onto slides and seal them.
*[[retinoic acid]]
*[[Retinol]]
*[[TTNPB]]
*[[BMS-453]]
 
===Shh===
*[[cyclopamine]]
*[[purmorphamine]]
*[[Vismodegib]]
 
===TGF-beta/BMP===
*[[SB-431542]]
*[[SB505124]]
 
===Thyroid Signaling===
*[[PTU]]
 
===Wnt===
*[[LiCl]]
*[[GSK3-inhibitor IX (BIO)]]
*[[IWR1]]
 
===Epigenetics===
*[[Ex-527]]
 
===Protease Inhibitors===
*[[chymostatin]]
*[[GM6001|GM6001 (MMP inhibitor)]]
*[[o-phenanthroline|o-phenanthroline(MMP inhibitor)]]
*[[BB-94|BB-94 MMP inhibitor]]
*[[GI254023X| GI254023X MMP10 inhibitor]]
 
==Small Molecules affecting Biological Functions & Processes==
===Angiogenesis===
*[[dimethyloxallyl glycine (DMOG)]]
*[[herbimycin A]]
*[[Suramin]]
 
===Apoptosis===
*[[cycloheximide]]
*[[alpha-amanitin]]
*[[Cyclosporin A]]
*[[2,2ʹ-Methylenebis(1,3-cyclohexanedione)]]
*[[NS3694]]
 
===Autophagy===
*[[LY-294002]]
 
===Calcium Signaling===
*[[nifedipine]]
*[[R(+)BayK 8644]]
*[[nicardipine]]
*[[deltamethrin]]
*[[BHQ]]
*[[Thapsigargin]]
 
===Circadian Rhythm===
*[[quinpirole]]
 
===Cytoskeletal Inhibitors===
*[[Cytochalasin B]]
*[[cytochalasin D]]
*[[Colcemide]]
*[[jasplakinolide]] is an activator
*[[latrunculin B]]
*[[taxol]]
*[[taxol|Paclitaxel]]
 
===Cell Cycle, including mitosis===
*[[Colcemide]]
*[[Hydroxyurea]]
*[[olomoucine]]
*[[PD-407824]]
===Cilia===
*[[methylcellulose (MC)]]
 
===DNA Damage/Repair===
*[[etoposide]]
*[[Hydroxyurea]]
 
===DNA Synthesis===
*[[Calyculin A]]
 
===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]]
 
===Hatching===
*[[ouabain]]
 
===Left-Right Patterning===
*[[methylcellulose (MC)]]
 
===Metamorphosis===
*[[3,3,5-triiodothyronine (T3)]]
*[[methimazole]]
*[[propylthiouracil]]
*[[thyroxine (T4)]]
 
===Neurotransmission===
*[[diazepam]]
*[[p-MPPI]]
*[[phaclofen]]
*[[RS67333]]
*[[deltamethrin]]
 
===Protein Recycling===
*[[primaquine]]
 
==Drugs by Gene Class==
 
===Channels, including ATPases===
*[[ouabain]]
*[[AM251]]
*[[AM281]]
*[[LY320135]]
===G Protein Receptors===
*[[purmorphamine]]
===Kinases===
*[[KT 5720]]
*[[olomoucine]]
*[[PD-407824]]
*[[SB 203580]]
*[[Y27632]]
*[[ZM 447439]]
 
===Phosphatases===
*[[Cyclosporin A]]
*[[deltamethrin]]
*[[okadaic acid]]
*[[tautomycin]]
*[[Calyculin A]]
 
===Small GTPases===
 
 
===Protein Synthesis===
*[[anisomycin]] 40uM Sigma
*[[cycloheximide]] 40uM
 
===Retinoids===
*[[TTNPB]] Sigma
 
==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]]
 
==Models of Anemia==
*[[Phenylhydrazine]]

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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