Goat anti eea1

Manufactured by Santa Cruz Biotechnology

Sourced in United Kingdom

Goat anti-EEA1 is an antibody product developed by Santa Cruz Biotechnology. It is designed to recognize the Early Endosomal Antigen 1 (EEA1) protein, which is a marker for early endosomes.

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10 protocols using goat anti eea1

Immunostaining of Human Islet Cells

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Human islets were prepared from beating heart donors with appropriate ethical permission and consents as described previously (54 (link)). Islets were dissociated into single cells and, after fixation in 4% (w/v) paraformaldehyde, were treated with antibodies as below (55 (link)). Rabbit anti-TPC2 antibody (1:150) was revealed with Alexa 568-conjugated secondary antibody (1:1500, Invitrogen, Paisley, UK). Guinea pig anti-insulin (1:300, DAKO, Ely, UK), goat anti-EEA1 (1:150, Santa Cruz Biotechnology, Santa Cruz, CA), and rat anti-LAMP-1 (1:150, Santa Cruz Biotechnology) were revealed with Alexa 488 secondary antibodies (1:1500, Invitrogen). Murine MIN6 clonal β cells (56 (link)) were transfected with plasmid encoding TPC2-mCherry using Lipofectamine 2000, and 48 h later were fixed, stained, and imaged as above. Images were captured using a Zeiss Axiovert 200 M spinning disc confocal imaging system (×40 oil immersion objective corrected for chromatic aberration; Hamamatsu ImageEM 9100-13 back-illuminated EM-CCD camera) with illumination (491 and 568 nm) provided by solid state lasers (Crystal Laser, NV) using a laser merge module (Spectral Applied Physics, Ontario, Canada) (57 (link)).

Arredouani A., Ruas M., Collins S.C., Parkesh R., Clough F., Pillinger T., Coltart G., Rietdorf K., Royle A., Johnson P., Braun M., Zhang Q., Sones W., Shimomura K., Morgan A.J., Lewis A.M., Chuang K.T., Tunn R., Gadea J., Teboul L., Heister P.M., Tynan P.W., Bellomo E.A., Rutter G.A., Rorsman P., Churchill G.C., Parrington J, & Galione A. (2015). Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Endolysosomal Two-pore Channels Modulate Membrane Excitability and Stimulus-Secretion Coupling in Mouse Pancreatic β Cells. The Journal of Biological Chemistry, 290(35), 21376-21392.

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Antibody Immunofluorescence Assay Protocol

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Primary antibodies used for indirect immunofluorescence were as follows: for Figures 1, 4, S5, and 6 we used rat anti-ATP7B (S. Lutsenko, Johns Hopkins School of Medicine); For Figures 2, S3, S4, 5, 7 & 8 we used rabbit anti-ATP7B (#ab124973, Abcam, Cambridge, MA); for Figures 3 and S1 we used rabbit anti-ATP7B (Dr. J. Gitlin, Brown Alpert Medical School, Providence, RI); rabbit anti-aminopeptidase N (APN, #1637, (22 (link)) guinea pig anti-dipeptidyl-peptidase 4 (DPP4, (41 (link))) goat anti-EEA1 (#sc-6415, Santa Cruz Biotechnology, Santa Cruz, CA); mouse anti-TGN38, and -Syntaxin 6 (BD Biosciences, San Jose, CA); mouse anti-LAMP1 (#H4A3-s, Developmental Studies Hybridoma Bank, Iowa City, IA); rabbit anti-Cathepsin D (Dr A. Hasilik, Marburg, Germany); rabbit anti-LC3B (#2775, Cell Signaling Technology, Danvers, MA). Secondary antibodies conjugated to Cy3 or Cy5 were from Jackson ImmunoResearch Laboratories (West Grove, PA), while those conjugated to Alexa 488, -568 or -647 were from Molecular Probes (Eugene, OR). Primary antibodies used for immunoblotting inculded: rabbit anti-ATP7B (#3985) (13 (link)), mouse anti-alpha tubulin, DM1A (Sigma, St. Louis, MO). Secondary antibodies conjugated to horseradish peroxidase (HRP) were from G.E. Healthcare (Buckinghamshire, United Kingdom).

Nyasae L.K., Schell M.J, & Hubbard A.L. (2014). Copper Directs ATP7B to the Apical Domain of Hepatic Cells via Basolateral Endosomes. Traffic (Copenhagen, Denmark), 15(12), 1344-1365.

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Fluorochrome-Conjugated Antibodies for Immunostaining

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Fluorochrome-conjugated antibodies used: anti-CD11c-APC, anti-IFNγ-APC, anti-TNFα-PE-cy7, anti-IL-4-PE, anti-IL17-PE, anti-Foxp3-PE (e-Bioscience, Vienna, Austria), and anti-LAMP-1-v450 (BD-Pharmingen). Unconjugated mouse anti-OVA (Sigma Aldrich), mouse anti-Le^X (Calbiochem), rat anti-mMGL (ER-MP23; kind gift from Dr. P. Leenen, Erasmus MC, Rotterdam, The Netherlands), rat anti-LAMP1 (BD-Pharmingen), rabbit anti-Rab11 (Life Technologies), goat anti-EEA1 (Santa Cruz Biotechnology) and rabbit anti-EEA-1 (Dianova). Secondary antibodies used: peroxidase-labeled F(ab’)₂ fragment goat anti-human IgG, F(ab’)₂ fragment goat anti-mouse IgG, (Jackson), peroxidase-labeled goat anti-mouse IgM (Nordic Immunology), goat anti-rat Alexa 448, goat anti-rat Alexa 647, donkey anti-goat Alexa 488, donkey anti-goat Alexa 647, donkey anti-rabbit Alexa 555 and donkey anti-rabbit Alexa 488 (Molecular Probes). MGL-1-Fc was generated as described earlier (Singh et al., 2009b (link)). MR-Fc was kindly provided by L. Martinez-Pomares (University of Nottingham, Nottingham, UK).

Streng-Ouwehand I., Ho N.I., Litjens M., Kalay H., Boks M.A., Cornelissen L.A., Kaur Singh S., Saeland E., Garcia-Vallejo J.J., Ossendorp F.A., Unger W.W, & van Kooyk Y. (2016). Glycan modification of antigen alters its intracellular routing in dendritic cells, promoting priming of T cells. eLife, 5, e11765.

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Antibody Immunolabeling Techniques

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The antibodies used in this study were goat anti-EEA1 (Santa Cruz Biotechnology, sc-6415), rabbit anti-megalin (kindly provided by Michele Marino, University of Pisa, Italy), mouse anti-acetylated-tubulin (Sigma, T7451), rabbit anti-endofin (Proteintech Europe, 13118-2-AP), mouse 3G8 and α6F anti-NaK ATPase (Developmental Studies Hybridoma Bank). Sheep anti-GFP antibodies were raised in-house. Fluorophore-conjugated secondary antibodies were purchased from Molecular Probes.

Oltrabella F., Pietka G., Ramirez I.B., Mironov A., Starborg T., Drummond I.A., Hinchliffe K.A, & Lowe M. (2015). The Lowe Syndrome Protein OCRL1 Is Required for Endocytosis in the Zebrafish Pronephric Tubule. PLoS Genetics, 11(4), e1005058.

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Immunofluorescence and Immuno EM Protocol

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Immunofluorescence: Mouse anti-syndecan-4 (1:50 dilution, 5G9, SC-12766), goat anti-EEA1 (1:200 dilution) and goat anti-Lamin A/C (1:50 dilution, N-19) were from Santa Cruz Biotechnologies Inc. (Santa Cruz, CA, USA). Rabbit anti-GM130 (1:200 dilution) and rabbit anti-desmin (1:80 dilution) were from Abcam (Cambridge, UK). Mouse anti-HA antibody (1:100 dilution,), Alexa 488 goat anti-mouse, Alexa 546 goat anti-mouse, Alexa 488 goat anti-rabbit and Alexa 647-conjugated donkey anti-goat were from Invitrogen (Carlsbad, CA, USA). DyLight 549-conjugated mouse anti-rabbit were from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA). DAPI was from Molecular probes (Invitrogen, Paisley, UK). Immuno EM: Mouse anti-HA (12CA5, 1:250 dilution) was from Life science Roche (Penzberg, Germany), and rabbit anti-mouse (1:175 dilution) was from Cappel Research Reagents (ICN Biochemicals, Irvin, CA, USA).

Rønning S.B., Carlson C.R., Stang E., Kolset S.O., Hollung K, & Pedersen M.E. (2015). Syndecan-4 Regulates Muscle Differentiation and Is Internalized from the Plasma Membrane during Myogenesis. PLoS ONE, 10(6), e0129288.

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Antibodies for Cell Signaling Analysis

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Rabbit anti-MAVS antibody was obtained from Bethyl Laboratories. Mouse anti-HA antibody, mouse anti-V5, goat anti-EEA1 and rabbit anti-GAPDH HRP-conjugated antibodies were purchase from Santa Cruz Biotechnology. Mouse anti-VSVG (P5D4) and mouse anti-DENV (clone D3-2H2-9-21) antibodies were purchased from Santa Cruz Biotechnology and Millipore, respectively. Alexa Fluor 488 or 594 phalloidin and Alexa fluor-conjugated secondary antibodies were purchased from Invitrogen.

Shu Q., Lennemann N.J., Sarkar S.N., Sadovsky Y, & Coyne C.B. (2015). ADAP2 Is an Interferon Stimulated Gene That Restricts RNA Virus Entry. PLoS Pathogens, 11(9), e1005150.

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Plasmid Construction and Antibody Verification

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Plasmids encoding GFP-tagged WT or mutant versions of human Arf1, Arfrp1, Arf1, Arl1, Arl14, and Sar1A were cloned into pEGFP-N1 vector. Plasmids encoding the WT or mutant versions of human Arfrp1 and Arl14 fused to FLAG and an IgG-binding ZZ domain were generated by cloning into the modified pcDNA4/TO vector encoding FLAG tag and ZZ domain (kindly provided by the Zhong laboratory, Shanghai Jiaotong University, Shanghai, China). Human Sys1 with 3× HA tag at its C terminus was synthesized by BGI (Beijing, China) in pcDNA3.1 vector. The antibodies used in this study were as follows: mouse anti–GFP (Roche, catalog no. 11814460001, RRID:AB_390913); sheep anti-TGN46 (Bio-Rad catalog no. AHP500G, RRID:AB_323104); rabbit anti-Sys1 (Thermo Fisher Scientific catalog no. PA5-48935, RRID:AB_2634391); goat anti-EEA1 (Santa Cruz, catalog no. sc-6415; RRID:AB_2096822); mouse anti-Myc (Cell Signaling Technology catalog no. 2276, RRID:AB_331783); and rabbit anti-HA (Cell Signaling Technology catalog no. 3724, RRID:AB_1549585). The siRNA target sequence against Sys1 is TCTCCATGATGTCCTTCAT.

Yang F., Li T., Peng Z., Liu Y, & Guo Y. (2021). The amphipathic helices of Arfrp1 and Arl14 are sufficient to determine subcellular localizations. The Journal of Biological Chemistry, 295(49), 16643-16654.

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Protein Extraction and Western Blot Analysis

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Cells were lysed in RIPA extraction buffer supplemented with 1 mM dithiothreitol (DTT) and 1X Protease Inhibitor Cocktail (Roche, Basel, Switzerland). Protein concentrations of cleared lysates were measured using the BCA assay before an equal amount of protein was loaded on precast 4-20% Tris/Glycine/SDS polyacrylamide gradient gels (BioRad). Transferred PVDF membranes were blotted using antibodies against: 1:100 rabbit anti-HACL1
(GeneTex, Irvine, CA; GTX106858), 1:4000 mouse anti-GAPDH (Santa-Cruz Biotechnology, Dallas, TX; sc-47724), 1:250 polyclonal rabbit anti-RBSN (Sigma-Aldrich, St. Louis, MO; HPA044878), 1:100 rabbit anti-PI3K (Proteintech, Rosemont, IL; 20584-1-AP), 1:100 goat anti-EEA1 (Santa-Cruz; sc-6415), 1:55 rabbit anti-CLTA (Sigma-Aldrich; HPA050918), 1:200 anti-CTSD (GeneTtex; GTX62063, Abcam; ab6313, Santa-Cruz; sc-6486), 1 µg/ml mouse anti-TFRC (Life Technologies / Thermofisher Scientific; 13-6800). Secondary anti-mouse-HRP, anti-goat-HRP and anti-rabbit-HRP were used at 1:4000 before visualization on x-ray film with SuperSignal West Dura Chemiluminescent Substrate (ThermoFisher Scientific).

Paul F., Ng C., Mohamad Sahari U.B., Nafissi S., Nilipoor Y., Tavasoli A.R., Bonnard C., Wong P.M., Nabavizadeh N., Altunoğlu U., Estiar M.A., Majoie C.B., Lee H., Nelson S.F., Gan-Or Z., Rouleau G.A., Van Veldhoven P.P., Massie R., Hennekam R.C., Kariminejad A, & Reversade B. (2022). RABENOSYN separation-of-function mutations uncouple endosomal recycling from lysosomal degradation, causing a distinct Mendelian disorder. Human molecular genetics, 31(21).

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Immunofluorescence Labeling of Primary Cells

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Primary cells were grown on a Nunc TM Lab-Tek TM 4-well glass chamber slide and fixed for 15 min in ice cold 4% (w/v) paraformaldehyde. Permeabilization using 0.6% (v/v) Triton-X in PBS was performed for 15 min, and then incubated with 1:55 rabbit anti-HACL1 (Abnova, Taipei, Taiwan; HPA055838), 1:67 rabbit anti-RBSN (Sigma-Aldrich; HPA044878), 1:50 goat anti-EEA1 (Santa-Cruz Biotechnology; sc-6415), 1 µg/ml mouse anti-RAB7 (Abcam, Cambridge, UK; ab50533); or 1:200 anti-CTSD (GenetTex; GTX62063) overnight at 4°C in 2% (w/v) BSA diluted with PBS. For visualization, 1:1000 secondary antibody conjugated to Alexa Fluor 568 or Alexa Fluor 488 (Molecular probes, Eugene, OR) was applied.
Counter staining of nuclei was performed using 1X Hoechst. Images were captured using the MetaMorph TM software on the FV1000 Olympus confocal microscope equipped with a Leica camera and lens.

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Generating Antibodies for Zebrafish Studies

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Polyclonal antibodies to zebrafish Pacsin2 were generated in sheep by immunising with a recombinant GST-tagged Pacsin2 construct encoding amino acids 301-388. Immunisation and serum collection were by Orygen Antibodies Ltd. Antibodies were affinity purified from serum by first clearing on GST beads alone followed by affinity purification on the GST-Pacsin2 recombinant protein. Polyclonal antibodies to zebrafish megalin were generated in rabbits against a GST fusion to the cytoplasmic domain, and affinity purified on the recombinant protein. Also used in this study were goat anti-EEA1 (Santa Cruz Biotechnology, sc-6415), mouse anti-Rab11 (BD Transduction Labs, 610657), mouse 3G8 anti-proximal tubule (European Xenopus Resource Centre, Portsmouth, UK), and mouse anti-GAPDH (Santa Cruz Biotechnology, sc-25778). Fluorophore-and HRP-conjugated secondary antibodies were purchased from Thermo Fisher Scientific.

Morgan J., Yarwood R., Starborg T., Yan G, & Lowe M. (2022). Pacsin2 is required for endocytosis in the zebrafish pronephric tubule. Biology Open, 11(6), bio059150.

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