Ab31261

Manufactured by Abcam

Sourced in United States, United Kingdom

Ab31261 is an antibody that can be used for immunodetection. It is a primary antibody that targets a specific antigen.

Automatically generated - may contain errors

Lab products found in correlation

Ab30687, by Abcam (3 mentions) Ab167453, by Abcam (2 mentions) Ab30637, by Abcam (2 mentions) Mab22591, by R&D Systems (2 mentions) Lsm 880, by Zeiss (1 mentions) Cy3 conjugated anti hrp, by Jackson ImmunoResearch (1 mentions) Axio observer z1 invert confocal microscope, by Zeiss (1 mentions) A11122, by Thermo Fisher Scientific (1 mentions) A11120, by Thermo Fisher Scientific (1 mentions) Rhodamine phalloidin, by Merck Group (1 mentions) Heptane, by Merck Group (1 mentions) Fv1000 confocal microscope, by Olympus (1 mentions) Tcs sp5 2, by Olympus (1 mentions) Ma5 15738, by Thermo Fisher Scientific (1 mentions) Dabco mowiol medium, by Merck Group (1 mentions) G10362, by Thermo Fisher Scientific (1 mentions) Anti laminc, by Developmental Studies Hybridoma Bank (1 mentions) Anti α tubulin, by Developmental Studies Hybridoma Bank (1 mentions) Anti β tubulin, by Developmental Studies Hybridoma Bank (1 mentions) T6557, by Merck Group (1 mentions)

10 protocols using ab31261

Larval Neuromuscular Junction Analysis

Cited 2 times

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Third instar wandering larvae were dissected, fixed, antibody stained, imaged and analysed as described previously (West et al., 2015 (link)). All NMJ analysis was performed double-blind. Primary antibodies used were Cy3-Conjugated anti-HRP (Goat, 1:200, Jackson ImmunoResearch Labs Cat^# 123–165-021, RRID:AB_2338959), anti-synaptotagmin (Rabbit, 1:2000, Syt-91, RRID:AB_2713991, (West et al., 2015 (link))) anti-polyubiquitinated proteins (Mouse, 1:2000, FK2, Enzo Life Sciences Cat^# BML-PW8810–0500, RRID:AB_2051891), anti-RAB4 (Rabbit 1:100, Abcam Cat^# ab78970, RRID:AB_2042753), anti-RAB5 (Rabbit, 1:500, Abcam Cat^# ab31261, RRID:AB_882240) and anti-Spinster (Guinea Pig, 1:1000, RRID:AB_2833057, (Sweeney and Davis, 2002 (link))). Drosophila motor-neurons were labelled using GFP-tagged even-skipped (eve). Confocal microscopy was performed using a Zeiss LSM 880 on an Axio Observer.Z1 invert confocal microscope (Zeiss). Z-stacked projections of NMJs and VNCs were obtained using a Plan Neofluar 40×/0.75 NA oil objective. NMJ lengths were measured from stacked NMJ images using the NeuronJ plugin for ImageJ (National Institutes of Health) as described previously (West et al., 2015 (link); West et al., 2018 (link)).

West R.J., Ugbode C., Fort-Aznar L, & Sweeney S.T. (2020). Neuroprotective activity of ursodeoxycholic acid in CHMP2BIntron5 models of frontotemporal dementia. Neurobiology of Disease, 144, 105047.

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Comprehensive Immunofluorescence Staining Protocol

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The following antibodies and concentrations were used for immunofluorescence staining: Armadillo 1:10 [mouse, N27A1, Developmental Studies Hybridoma Bank (DSHB)], Arrow 1:10,000 (rabbit, a kind gift from Steve DiNardo, University of Pennsylvania, USA), Calnexin 1:10 (mouse, Cnx99A 6-2-1, DSHB), DE-cadherin 1:5 (rat, DCAD2, DSHB), Distalless 1:250 (rabbit, a kind gift from Grace Boekhoff-Falk, University of Wisconsin-Madison, USA), GFP 1:500 (mouse, A11120, and rabbit, A11122, Molecular Probes), GM130 1:250 (rabbit, ab30637, Abcam), Hrs 1:10 (mouse, Hrs8-2 and Hrs27-4, DSHB), Lamp1 1:100 (rabbit, ab30687, Abcam), mCherry 1:1000 (rabbit, ab167453, Abcam), Rab5 1:500 (rabbit, ab31261, Abcam), Rab7 1:10 (mouse, Rab7, DSHB), Sens [rabbit 1:1000, a kind gift from Hugo Bellen (Howard Hughes Medical Institute, Houston, TX, USA)], α-tubulin 1:100 (mouse, 12G10, DSHB), Vestigial 1:100 (rabbit, a kind gift from Kirsten A. Guss, Dickinson College, USA), Wg used 1:3 for extracellular and 1:20 for total staining (mouse, 4D4, DSHB) and Evi 1:500 [rabbit, a kind gift from Konrad Basler (University of Zurich, Switzerland)]. Secondary antibodies directed against the species of interest were coupled to Alexa Fluor 488, 568 and 647 (1:500, Invitrogen).

Witte L., Linnemannstöns K., Schmidt K., Honemann-Capito M., Grawe F., Wodarz A, & Gross J.C. (2020). The kinesin motor Klp98A mediates apical to basal Wg transport. Development (Cambridge, England), 147(15), dev186833.

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Immunostaining of Ovarian Tissue

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Ovary dissection was carried out in phosphate-buffered saline (PBS) and then fixed in Devitellinizing buffer (7% formaldehyde) and heptane (Sigma) mixture (1:6) for 10min. After washes in PBS, ovaries were incubated in blocking solution (PBT, 10% goat serum) for 30min and then stained overnight at 4 °C. Primary antibodies and their concentrations were as follows: mouse anti-Arm (1:50; N2 7A1; Developmental Studies Hybridoma Bank (DSHB)), rabbit anti-PKCζ (1:200; C-20; Santa Cruz), mouse anti-Crb (1:10; Cq4; DSHB), rat anti-Ncad (1:20; DN-Ex #8; DSHB), mouse anti-β-tubulin (1:500; E7; DSHB), rabbit anti-Baz (1:400; kind gift from A. Wodarz), mouse anti-Dlg (1:50; 4F3; DSHB), rabbit anti-Sdt (1:500)38 (link), rabbit anti-PATJ (1:1000)39 (link), rabbit anti-Lva (1:200)40 (link), rabbit anti-Rab5 (1:100, ab31261, Abcam), rabbit anti-Rab7 (1:3000)41 (link) and rabbit anti-Rab11 (1:8000)41 (link). Methanol treatment was used after fixation for anti-Crb staining. After washes in PBT, ovaries were incubated with secondary antibodies (1:250, Jackson ImmunoResearch) for 2 hours at room temperature. F-actin was labeled by Rhodamine phalloidin (1:100, Sigma). Confocal images were obtained using a Leica TCS SP5 II or an Olympus FV1000 confocal microscope.

Luo J., Wang H., Kang D., Guo X., Wan P., Wang D, & Chen J. (2016). Dlg5 maintains apical polarity by promoting membrane localization of Crumbs during Drosophila oogenesis. Scientific Reports, 6, 26553.

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Production and Validation of Antibodies

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Purified Tsf1 (0.3 mg) was sent to Cocalico Biologicals for the production of polyclonal antiserum in a guinea pig. We used commercially available rabbit antiserum against D. melanogaster Rab5 (Abcam #ab31261). This antiserum has been used by others to identify endosomes in neural tissue, wing discs, eye discs, and other tissues (Fan et al., 2013 (link); Yuva-Aydemir et al., 2011 (link); Zschätzsch et al., 2014 (link)). A mouse monoclonal antibody that recognizes D. melanogaster β-tubulin (Hybridoma Product E7) was purchased from the Developmental Studies Hybridoma Bank at the University of Iowa. E7 was developed by M. McCutcheon and S. Carroll and was deposited by M. Klymkowsky. A mouse monoclonal antibody that recognizes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purchased from Thermo Fisher (#MA5-15738).

Weber J.J., Brummett L.M., Coca M.E., Tabunoki H., Kanost M.R., Ragan E.J., Park Y, & Gorman M.J. (2022). Phenotypic analyses, protein localization, and bacteriostatic activity of Drosophila melanogaster transferrin-1. Insect biochemistry and molecular biology, 147, 103811.

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Comprehensive Antibody Characterization Protocol

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Antibodies were used against human Alix (1:100 WB; goat, Santa Cruz and mouse, Santa Cruz), ɑ‐Tubulin (1:100 WB; mouse, DSHB 12G10), human ATP6V0A1, (1:1000 WB; rabbit, Novus Bio), Cnx (1:10 IF; mouse, DSHB Cnx99A 6‐2‐1), Csp‐1(1:50 WB; mouse, DSHB ab49), Csp‐2(1:50 WB; mouse, DSHB 6D6), Flo2(1:1000 WB; BD Transduction Laboratories), GFP (1:1000 WB and IF; mouse (A11120) and rabbit (A11122), Molecular Probes), GM130 1:1000(WB; rabbit, Abcam ab30637), Golgin 1:10(IF; mouse DSHB Golgin84), Hrs (1:50 WB; 1:10 IF; mouse, DSHB Hrs 27‐4 and Hrs 8‐2), Hsp70(1:1000 WB; mouse Santa Cruz BRM22), (IntegrinβPS (1:50 WB; mouse, DSHB CF.6G11), Lamp1(1:500 WB; 1:100 IF; rabbit, Abcam ab30687), Lbm (1:50 WB; 1:10 IF; mouse, DSHB 10C9), mCherry (1:1000 IF and WB; rabbit, Abcam ab167453), Rab5(1:500 IF; rabbit, Abcam ab31261), Rab7(1:10 IF; mouse, DSHB), human Rab11(1:1000 WB; mouse, BD Transduction), Syx1A (1:50 WB; mouse, DSHB 8C3), Tsg101(1:500 WB; mouse, Abcam 4A10). Secondary antibodies directed against the species of interest were coupled to Alexa Fluor 488, 568 and 647(IF, 1:400, Invitrogen) and 680RD and 800CW (WB, 1:20,000, LiCor).

Linnemannstöns K., Karuna M P., Witte L., Choezom D., Honemann‐Capito M., Lagurin A.S., Schmidt C.V., Shrikhande S., Steinmetz L., Wiebke M., Lenz C, & Gross J.C. (2022). Microscopic and biochemical monitoring of endosomal trafficking and extracellular vesicle secretion in an endogenous in vivo model. Journal of Extracellular Vesicles, 11(9), 12263.

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Immunofluorescence Staining of Drosophila Tissues

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Tissues from third instar larvae were processed as described previously [5 (link)]. The following primary and secondary antibodies were used at the indicated dilutions: anti-Dlg1 (RRID: AB_528203; 1:200), anti-Fasciclin III (RRID: AB_528238; 1:200), anti-Elav (RRID: AB_528217 and AB_528217; 1:200), anti-DE-cad (RRID: AB_528120; 1:200), anti-Crumbs (RRID: AB_528181; 1:200), anti-p120-catenin (RRID: AB_2088073; 1:200), anti-α-Tubulin (RRID: AB_579793; 1:200), anti-β-Tubulin (RRID: AB_2315513; 1:200), anti-LaminC (RRID: AB_528339; 1:500) and anti-βPS integrin (RRID: AB_528310; 1:200) from the Developmental Studies Hybridoma Bank (DSHB) (Iowa City, Iowa), anti-γ-Tubulin (T6557, Sigma Aldrich; 1:50), anti-GFP (G10362, Invitrogen; 1:500), anti-Rab5 (ab31261, Abcam; 1:200), anti-Rab11 (BD610656, BD Biosciences; 1:200), chicken-anti-Avl (a gift from D. Bilder, 1:500) and Cy2, Cy3- and Cy5-conjugated secondary antibodies (Jackson Immunoresearch). Tissues were counterstained with DAPI (1 μg/ml) and mounted in DABCO-Mowiol medium (Sigma-Aldrich).

Donohoe C.D., Csordás G., Correia A., Jindra M., Klein C., Habermann B, & Uhlirova M. (2018). Atf3 links loss of epithelial polarity to defects in cell differentiation and cytoarchitecture. PLoS Genetics, 14(3), e1007241.

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Antibody Validation for Drosophila Studies

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The following antibodies were used in this study: Mouse anti-Drosophila Rab7 hybridoma supernatant (1:5) and goat anti-Drosophila golgin245 (both developed and validated by S. Munroe [Riedel et al., 2016 (link)] and obtained from the Developmental Studies Hybridoma Bank). Rabbit anti-Drosophila Rab5 (1: 250, Ab31261; Abcam). Rat anti-tdTomato (1:500, EST203; Kerafast). Mouse anti-GFP (1:500, 1181446000; Sigma-Aldrich). Mouse anti-insect cathepsin L antibody (1:500, MAB22591; R&D Systems). Mouse anti-tubulin antibody (1:2,000, T9026; Sigma-Aldrich). The guinea pig anti-Drosophila scattered (Vps54) antibody was generated by and obtained from R. Sinka (Fári et al., 2016 (link); 1:400). Secondary antibodies for immunohistochemistry were anti-mouse, goat, rabbit, or rat labeled by Alexa 488, 555, or 647 (1:1,000; Thermo Fisher Scientific). Secondary antibodies for Western blot were anti-mouse HRP (1:1,000, 115-035-146; Jackson), anti-guinea pig HRP (1:500, A5545; Sigma-Aldrich), or anti-mouse IR-Dye 680 LT (1:20,000; LI-COR).

O’Brien C.E., Younger S.H., Jan L.Y, & Jan Y.N. (2022). The GARP complex prevents sterol accumulation at the trans-Golgi network during dendrite remodeling. The Journal of Cell Biology, 222(1), e202112108.

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Antibodies and Chemicals for Lysosome Analysis

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The following commercially available antibodies were used: anti-Rab5 (ab31261, abcam), anti-GABARAP for Drosophila Atg8a (PM037, MBL), anti-Drosophila LAMP1 (ab30687, abcam), anti-human LAMP1 (AB2296838, DSHB), anti-cathepsin D (sc-6487, Santa Cruz), anti-Brp (AB2314866, DSHB), anti-cathepsin L (MAB 22591, R&D systems)^{44 (link),63}, anti-Ref(2)P²⁸, anti-elav (AB528217, DSHB), anti-ATP5α (ab14748, abcam), Cy5-conjugated anti-HRP (123175021, Jackson ImmunoLab), and secondary antibodies conjugated to Alexa 488/568/647 (Thermo Fisher Scientific), or near infrared (IR) dye 700/800 (Rockland). The following chemicals were used in the study: N-acetylcysteine amide (A0737, Sigma-Aldrich), N-(2-mercaptopropionyl)glycine (M6635, Sigma-Aldrich), putrescine (51799, Sigma-Aldrich), spermidine (S0266, Sigma-Aldrich), spermine (s4264, Sigma-Aldrich), bis(hexamethylene)triamine (421960, Sigma-Aldrich), DHE (D11347, Thermo Fisher Scientific), LysoTracker^® Red DND-99 (L7528, Thermo Fisher Scientific), LysoSensor™ Yellow/Blue DND-160 (L7545, Thermo Fisher Scientific).

Li C., Brazill J.M., Liu S., Bello C., Zhu Y., Morimoto M., Cascio L., Pauly R., Diaz-Perez Z., Malicdan M.C., Wang H., Boccuto L., Schwartz C.E., Gahl W.A., Boerkoel C.F, & Zhai R.G. (2017). Spermine synthase deficiency causes lysosomal dysfunction and oxidative stress in models of Snyder-Robinson syndrome. Nature Communications, 8, 1257.

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Immunostaining of Rab5 and Rab7 in Drosophila Larvae

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3rd instar larvae expressing handC driven Mon1::HA and Mon1^∆100::HA were dissected in PBS and fixed with 4% PFA in PBS. After washing, specimens were permeabilized with 1% Triton X-100 in PBS, followed by washing with BBT (0.1% BSA and 0.1% Tween-20 in PBS). Specimens were incubated for 30 min in blocking solution (1% BSA and 0.1% Tween-20 in PBS) followed by antibody staining (rabbit anti-Rab5, 1:250, Abcam Cat# ab31261, RRID:AB_882240, Cambridge, United Kingdom; mouse anti-Rab7, 1:10, Developmental Studies Hybridoma Bank Cat# Rab7, RRID:AB_2722471, University of Iowa, IA, USA) (52 (link)). Afterwards they were rinsed with BBT, blocked with blocking solution, and incubated with secondary antibodies (anti-rabbit Alexa Fluor 488, 1:200, Jackson ImmunoResearch Laboratories, Inc, Ely, United Kingdom, Code Number: 111-545-006); anti-mouse Cy3, 1:200, Dianova GmbH, Eching, Germany). Samples were embedded in Fluoromount-G mounting medium containing DAPI (Thermo Fisher, Waltham, MA, USA). Confocal images were captured with an LSM800 (Zeiss, Jena, Germany). For details, see SI Appendix.

Borchers A.C., Janz M., Schäfer J.H., Moeller A., Kümmel D., Paululat A., Ungermann C, & Langemeyer L. (2023). Regulatory sites in the Mon1–Ccz1 complex control Rab5 to Rab7 transition and endosome maturation. Proceedings of the National Academy of Sciences of the United States of America, 120(30), e2303750120.

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Multicolor Immunofluorescence of Drosophila Brain

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Brains from synchronized, staged animals were dissected in phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde in PBS for 30 min at room temperature (RT), and then blocked for 1 h at RT in PBS with 0.2% Triton X-100 (PBST) + 1% bovine serum albumin. Primary antibodies were diluted in the blocking buffer for incubation overnight at 4 °C, followed by washing 3X with PBST. Secondary antibodies were incubated for 3 h at RT and then washed 3X with PBST. Primary antibodies used were mouse anti-PDF [Developmental Studies Hybridoma Bank (DSHB), PDF C7,1:50], rabbit anti-Repo (gift from Dr. Benjamin Altenhein, University of Cologne, Germany, 1:1,000), chicken anti-GFP (Abcam, ab13970, 1:1,000), rabbit anti-Rab5 (Abcam, ab31261, 1:500), rabbit anti-pAkt (phospho S473, Cell Signaling, 4060S, 1:100), and mouse anti-Rab7 (DSHB, Rab7, 1:100). Secondary antibodies used were goat anti-mouse 488 (Invitrogen, A11001, 1:250), donkey anti-rabbit 555 (Invitrogen, A31572, 1:250), goat anti-chicken 488 (Invitrogen, A11039, 1:250), and goat anti-mouse 633 (Invitrogen, A21050, 1:250).

Song C, & Broadie K. (2023). Fragile X mental retardation protein coordinates neuron-to-glia communication for clearance of developmentally transient brain neurons. Proceedings of the National Academy of Sciences of the United States of America, 120(12), e2216887120.

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