SDS-PAGE gels (Conlon 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 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.


link to protocol document [http://www.unc.edu/~fconlon/Protocols/SDS_page.docx]
==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]


==Alphabetical List of Small Molecules With Literature References==


CASTING A SDS-PAGE (DENATURING) GEL
*[[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]]


'''IMPORTANT Clean glass spacers and plates prior to and IMMEDIATELY after use! Failure to do so will result in uneven gel preparation, cracking of glass plates, and leaking of gel before polymerization. '''
==Small Molecules Affecting Pathways==
===cAMP===
*[[IBMX]]
*[[RU486]]


1. Clean sufficient amount of glass spacers and plates for your experiment (If you are running two gels, clean two spacers and two plates). Clean first with 95% ethanol and wipe clean with kimwipe, then clean with distilled water, wipe clean. Allow to air-dry sufficiently on paper towel until ready to cast gel.
===CDK/Cyclins===
*[[olomoucine]]
*[[RO-3306]]


2. Prepare stacking and resolving gel solutions, minus the APS and TEMED, which polymerize the gel. For protein gel applications, we use Bio-Rad bisacrylamide #161-0148, pore size 37.5:1.  BE SURE TO CHECK % of stock solution before using.  The % resolving  gel you use should be determined by the sizes of proteins you are attempting to resolve. 7.5% is better for viewing larger proteins, 120kDa and up.  15% is better for viewing smaller proteins ( i.e. <50 kDa). The stacking gel is fixed at 5% acrylamide. This allows all the proteins in your sample to run evenly into the resolving gel.
===ERK/MAPK===
*[[U0126]]
===Folic Acid===
*[[Methotrexate (MTX)]]


:CAUTION: Bisacrylamide in liquid form is really bad for you. Use extreme caution to make sure you don’t spill it, especially on yourself. Once polymerized to solid state, it is no longer a danger.
===JAK/STAT===
*[[AZD1480]]


===JNK===
*[[SP600125]]
*[[Pyrazoleanthrone]]


Recipe for gels (adjust water and acrylamide for 40% stock):
===Nitric Oxide===
*[[sodium nitroprusside]]


Resolving  Gels (15 ml) 
===PDGF===
{|border="1" cellpadding="5"
*[[AG1296]]
|-
*[[AP1510]]
|
! scope="col" style="background:#efefef;" | 7.50%
! scope="col" style="background:#efefef;" | 10%
! scope="col" style="background:#efefef;" | 12%
! scope="col" style="background:#efefef;" | 15%
|-
! scope="row" style="background:#efefef;" | 1.5M Tris pH 8.8
|5 ml
|5 ml
|5 ml
|5 ml
|-
! scope="row" style="background:#efefef;" | 30% acrylamide
|3.75 ml
|4 ml
|6 ml
|6 ml
|-
! scope="row" style="background:#efefef;" | water
|6.15 ml
|5.9 ml
|3.9 ml
|2.4 ml
|-
! scope="row" style="background:#efefef;" | 20% SDS
|75 ul
|75 ul
|75 ul
|75 ul
|-
! scope="row" style="background:#efefef;" | 10% APS
|75 ul
|75 ul
|75 ul
|75 ul
|-
! scope="row" style="background:#efefef;" | TEMED
|25 ul
|25 ul
|25 ul
|25 ul
|}


===Retinoic Acid===
*[[AGN193109]]
*[[Citral]]
*[[retinoic acid]]
*[[Retinol]]
*[[TTNPB]]
*[[BMS-453]]


Stacking Gels (5 ml)
===Shh===
*[[cyclopamine]]
*[[purmorphamine]]
*[[Vismodegib]]


{|border="1" cellpadding="5"
===TGF-beta/BMP===
|-
*[[SB-431542]]
! scope="row" style="background:#efefef;" |0.5M Tris pH 6.8
*[[SB505124]]
|.62 ml
|-
! scope="row" style="background:#efefef;" |30% acrylamide
|.833 ml
|-
! scope="row" style="background:#efefef;" |0.5M Tris pH 6.8
|.62 ml
|-
! scope="row" style="background:#efefef;" |water
|3.817 ml
|-
! scope="row" style="background:#efefef;" |20% SDS
|25 ul
|-
! scope="row" style="background:#efefef;" |10% APS
|50 ul
|-
! scope="row" style="background:#efefef;" |TEMED
|5 ul
|}


===Thyroid Signaling===
*[[PTU]]


These recipes can make up to 4 gels – making this much for 2-3 gels is not a bad idea. This way you ensure you have enough solution in case you have leakage issues.
===Wnt===
*[[LiCl]]
*[[GSK3-inhibitor IX (BIO)]]
*[[IWR1]]


3. Assemble gel spacers and holders in mini-gel casting apparatus.
===Epigenetics===
*[[Ex-527]]


4. Add 10% APS and TEMED to Resolving Gel Solution and mix well.  Using P1000, add  1 ml solution at a time in between assembled spacer and plate.  Leave about ¾ inch space from top of the plate (or just fill to top green horizontal bar on gel holder).
===Protease Inhibitors===
*[[chymostatin]]
*[[GM6001|GM6001 (MMP inhibitor)]]
*[[o-phenanthroline|o-phenanthroline(MMP inhibitor)]]
*[[BB-94|BB-94 MMP inhibitor]]
*[[GI254023X| GI254023X MMP10 inhibitor]]


5. Immediately add 100% EtOH to overlay the resolving gel. This will remove any bubbles in the gel. EtOH does not mix with the acrylamide, so as the acrylamide polymerizes, the EtOH will remain atop the gel. Allow to polymerize at least 10 minutes.
==Small Molecules affecting Biological Functions & Processes==
===Angiogenesis===
*[[dimethyloxallyl glycine (DMOG)]]
*[[herbimycin A]]
*[[Suramin]]


6. Rinse off EtOH will copius amounts of distilled water. Ensure there is no water left on the resolving gel.
===Apoptosis===
*[[cycloheximide]]
*[[alpha-amanitin]]
*[[Cyclosporin A]]
*[[2,2ʹ-Methylenebis(1,3-cyclohexanedione)]]
*[[NS3694]]


7. Add 10% APS and TEMED to Stacking Gel Solution and mix well.  Using P1000, add  1 ml solution at a time until gel is completely full. AVOID BUBBLES!  Add appropriate gel combs to stacking gel immediately. Again, wait at least 10 minutes for gel to polymerize.
===Autophagy===
*[[LY-294002]]


8. Assemble gel in electrophoresis chamber. Fill inner chamber with running buffer. Remove gel combs, and fill inner chamber again. There should be no need to clear out wells, there should be no acrylamide or bubbles in them. Load gel and run. Recommended: Run 70V for about 20-30 min until sample has run through the stacking gel. At that point, voltage can be increased to 150V to 200V if needed. Better resolution will occur at lower voltages!
===Calcium Signaling===
*[[nifedipine]]
*[[R(+)BayK 8644]]
*[[nicardipine]]
*[[deltamethrin]]
*[[BHQ]]
*[[Thapsigargin]]


9. Once gel has finished running, gently pop glass plate off the spacer using the PLASTIC green spatula. Gently remove gel and transfer or Coomassie stain as normal.
===Circadian Rhythm===
*[[quinpirole]]


10. Make sure to clean glass plates well after use! A common practice is to soak them in HOT water while tending to immediate needs, such as transfer or staining. Do not leave them for long after you have run the gel, as residual acrylamide on plates will affect casting of subsequent gels. Be sure to clean off with hot water and then DI water to remove any salts. Place in drying rack.
===Cytoskeletal Inhibitors===
*[[Cytochalasin B]]
*[[cytochalasin D]]
*[[Colcemide]]
*[[jasplakinolide]] is an activator
*[[latrunculin B]]
*[[taxol]]
*[[taxol|Paclitaxel]]


Notes:
===Cell Cycle, including mitosis===
*[[Colcemide]]
*[[Hydroxyurea]]
*[[olomoucine]]
*[[PD-407824]]
===Cilia===
*[[methylcellulose (MC)]]


10% APS (Ammonium persulfate) should be made fresh and stored at 40C for up to ~2 weeks. Aliquots can be stored at -200C indefinitely. 
===DNA Damage/Repair===
*[[etoposide]]
*[[Hydroxyurea]]


TEMED smells bad (or so Erin says). Open and close quickly. Close tightly.
===DNA Synthesis===
*[[Calyculin A]]


Gels can be cast and left overnight at 4 degrees. To do so, wrap tightly with wet paper towels and again with plastic wrap to keep gel from drying. Keep in mind, this should be done minimally as the number of plates are limited.
===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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