antibodies that work in tribolium castaneum kevin burns 5/20/2011
TRANSCRIPT
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Antibodies that Work In Tribolium Castaneum
Kevin Burns5/20/2011
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Notes on this Document• For antibodies I’ve used personally, the titles contain the following information
– Original antigen used to produce antibody– A rough guess at the stage/stages of Tc shown– Antibody procurement info, e.g. DSHB, Supplier, or Lab– Host species and working dilution
• I have often thrown in co-stains to further illustrate morphology and to make things look cool. However, mostly this is just confusing.– KT817 is an enhancer trap line from Sue Brown that marks oenocytes. It is also cool for marking
A1-A8
• The second half of this document in-exhaustively covers antibodies that have been used in published works, normally on beetle, but occasionally on other arthropods, please update me if you find any new ones ([email protected])
• There is a PDF and a Powerpoint version of this document (The hyperlinks work in the PPT), if your email server does not support attachments of such & such size, I will post this document on http://burnska08.wordpress.com/kevin-burns-home-page/
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Index• AbdA (+UBX)• AntP• Futsch (22c10)• HNF4• Notch Intracellular Domain• Orthodenticle (GP / Rat)• Phospho-Histone3• Phospho-MAPK• Spalt• Trachea (a2A12)• UBX (see AbdA)
Index of Other’s Results
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Dm Abd-A/UBXPre-Hatching, ~64h at 30C
DSHB: Mouse Monoclonal, 1:5
UBX/AbdA UBX/AbdADAPI KT817-YFP
Index for KB’s Antibodies
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Dm AntPGermband retraction 30h @30C
DSHB: Mouse Monoclonal, 1:25+
NICD
AntP
DAPI
KT817-YFP
AntP
Please advise naïve young investigators that AntP is not just expressed in thoracic segments but also in the CNS Index for KB’s Antibodies
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Dm En/InvDorsal closure/Leg articulation (36h)
DSHB: 4D9Mouse Monoclonal, 1:25
En/InvSpalt En/Inv
Index for KB’s Antibodies
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Tc Engrailed
Cocalico/Gebelein, coming in JulyRat/Guinea Pig
Kevin Burns is making this in Brian Gebelein’s lab in order to do some co-stains with the multitude of mouse antibodies
Index for KB’s Antibodies
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Dm FutschDorsal closure/48h? (Ventral View)
DSHB: 22c10Mouse Monoclonal, 1:50
22c10
Index for KB’s Antibodies
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HNF448h @ 30C
Cocalico/Gebelein: Rat Serum, 1:1000
HNF4 HNF4KT817-YFP
p
Mesodermal staining (arrowheads), continues underneath more superficial oenocyte staining (e.g. red circle)p = pleuropod in A1 Index for KB’s Antibodies
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Notch Intracellular DomainGermband extension
DSHB: C17.9C6Mouse Monoclonal 1:5
NICD NICDDAPI
Index for KB’s Antibodies
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Dm OTD full lengthMix sample
Cocalico/Cook: Rat Serum, 1:500
OTD
I am conducting another stain to see if this detects the OTD that replaces bicoid in early patterning…
Index for KB’s Antibodies
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Dm OTD full lengthMix sample
Cocalico/Cook: Guinea Pig Serum, 1:500
OTD
Index for KB’s Antibodies
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phospho-Histone 3Full extension
Millipore: 05-806Mouse, 1:1000
pH3 pH3 DAPI
Index for KB’s Antibodies
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Di-Phosphorylated MAPKpost-gastrulation and full germband
Sigma: M-8159Mouse Monoclonal, 1:50
pERKpERK
Index for KB’s Antibodies
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Dm Spalt Major24h, 36h @ 30C
Cocalico/Gebelein: Rabbit Serum, 1:1500
SpaltSpalt
pERKSpalt
KT-817-YFP
Spalt
Index for KB’s Antibodies
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Dm unknown (tracheal System)Pre-HatchingDSHB: a2A12
Mouse IGM Monoclonal, 1:25
NICD
a2A12DAPI
This stain was achieved with IgG secondary antibodies, my guess is secondaries designed against IgM would fare much better
Index for KB’s Antibodies
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Table of Kevin’s Antibodies
Cat#/name Antigen Host Isotype Concentration Stains what Available From
4C3 Dm AntP M IgG 1:50Thoracic segments, ventral nervous system DSHB
22c10 Dm Futsch M IgG 1:50 CNS commissures only (so far) DSHB
HNF4 Tc HNF4 Rat Serum 1:2000Malpighian tubules, hindgut, fat bodies, oenocytes Brian Gebelein
FP6.87 Dm AbdA/Ubx M IgG 1:5
T3/Abdominal segments less those in AbdB domain, Recognizes both AbdA and Ubx DSHB
A2a12 Dm? M IgM 1:25 Tracheal system DSHBSpalt 26 Dm Spalt Rabbit Serum 1:1500 Ectodermal streak Brian Gebelein
4D9 Dm Engrailed M IgG 1:10+ Posterior segmental boundaries DSHB2B8 Dm Eve M DSHB
C17.9C6 Dm NICD M IgG11:5 better than 1:25 Notch Receiving Cells DSHB
M-8159 pErk M 1:50 Di-phosphorylated MAPK Sigma pH3 M 1:1000 easily Mitotic cells Millipore Caspase3 M Apoptotic cells Cell SignalingG6 Dm Otd Guinea Pig serum 1:500- Ventral CNS Tiffany CookR2 Dm Otd Rat serum 1:500- Ventral CNS Tiffany Cook
Index for KB’s Antibodies
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Index of Other People’s • Caudal • Caspase 3• Cx (scr)• DC0 (mushroom body etc) • DLL • engrailed 4F11 from DSHB• Even-skipped• Nubbin (Drosophila + Crustacean + Chelicerates) • Prd, Gooseberry, Gooseberry-Neuro (Pairberry)• pSMAD • pY• Synapsin 1
Index for KB’s Antibodies
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CaudalShulz&Tautz 1998 Dev Genes & Evolution
Generation of an antibodyPart of the cadA ORF (open reading frame, corresponding to aminoacids 79–249) was amplified by PCR and cloned in frame intothe EcoRV site of the pRSET-C (Invitrogen) vector. The
Index of Other’s Results
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Caspase 3Aranda&Tautz2009 Dev Genes Evol
For apoptosis detection using the anti cleaved caspase3 antibody (Cell Signalling Technology, Inc.)
Index of Other’s Results
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Cx (Scr)Shippy & Denell 2006 Genetics
A cross-reacting polyclonal antibody to Drosophila Scr, α-DmScr (a gift from Thomas Kaufman), was used to detect Cx
Index of Other’s Results
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DC0Zhao&Farris 2008
Anti-DC0 primary antibody, a polyclonal antibodyagainst the catalytic subunit of Drosophila melanogasterprotein kinase A, was a gift provided by Dr. Daniel Kalderon.This antibody has been shown to have a high affinityfor mushroom body intrinsic neurons in a range of insectspecies (Skoulakis et al., 1993; Farris, 2005a,b). Index of Other’s Results
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DLLe.g. Panganiban&Carroll 1997 PNAs
• Butterfly Staining…
Production and characterization of the Dll homeodomain antibody was described in Panganiban et al. (4)
Index of Other’s Results
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Engrailed 4F11 (alternate DSHB Clone)e.g. Pueyo&Couso2008 PNAS
This is cochroach, but I swear I’ve seen beetle people use 4f11 instead of 4D9
Index of Other’s Results
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Even SkippedBrown & Denell 1997
Choe & Brown 2006Grasshopper antigen, see also Patel 1994
Index of Other’s Results
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NubbinLi & Popadic 2004 Evolution& Development
A monoclonal antibody that recognizes a conserved epitope in the Nub/pdm protein has been generated and used successfully in a number of crustacean and chelicerate species (Averof and Cohen 1997; Abzhanov and Kaufman 2000; Damen et al. 2002). Unfortunately, this antibody does not recognize insect nub orthologs except for that of Drosophila. Index of Other’s Results
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Pairberry (Prd, Gsb, Gsbn)Davis & Patel 2001
Index of Other’s Results
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Phospho-SMADNunes da fonseca & Roth 2008 Dev Cell
Index of Other’s Results
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Phospho-TyrosineNunes da fonseca & Roth 2008 Dev Cell
Anti-phosphotyrosine antibody staining marks the cell outlines (red), DAPI (blue)
Index of Other’s Results
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Synapsin 1Dreyer&Schactner2010
The monoclonal primary antibody from mouse against a fusion protein consisting of a glutathione-S-transferase and the first amino acids of the presynaptic vesicle protein synapsin I coded by its 5 -end ′(SYNORF1, Klagges et al., 1996 )
Index of Other’s Results