Anti-PS1-N-terminus (1–65) (PRB-354P, 1:1,000, Covance, Davis, CA, USA); Anti-PS1-N-terminus (MAB1563, 1:100, EMD Millipore, Darmstadt, Germany); anti-PS1 loop (263–407) (529592, 1:2,000, Calbiochem); anti-APH1aL/C terminal (245–265) (PRB-550P, 1:1,000, Covance); anti-NCT (N1660, 1:1,000, Sigma), Anti-Pen2 (P5622, 1:1,000, Sigma); anti-BACE1 (AP7774b, 1:1,000, Abgent, Suzhou, China); anti-HA (H6908, 1:5,000, Sigma); anti-Flag (F3156, 1:2,000, Sigma); anti-A2AR (05–717, 1:1,1000, Millipore); anti-EEA1 (610457, dilution 1:200, BD transduction laboratories).
Anti eea1
Manufactured by BD
Sourced in United States, United Kingdom
Anti-EEA1 is a laboratory reagent used for the detection and analysis of the Early Endosome Antigen 1 (EEA1) protein. EEA1 is a marker for early endosomes, which are important organelles involved in the intracellular trafficking and sorting of molecules. The Anti-EEA1 reagent can be used in various cell and tissue analysis techniques, such as immunofluorescence, Western blotting, and immunohistochemistry, to visualize and quantify the presence and distribution of EEA1 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti lamp1, by Abcam (4 mentions)
Lsm 880 confocal microscope, by Zeiss (4 mentions)
Anti flag, by Merck Group (3 mentions)
Anti gm130, by BD (3 mentions)
Anti cd63, by Santa Cruz Biotechnology (3 mentions)
Anti giantin, by Abcam (2 mentions)
Anti lamp1, by BD (2 mentions)
Anti rab7, by Santa Cruz Biotechnology (2 mentions)
Anti glt1, by BD (2 mentions)
Alexa fluor 488 or 555 or 647 conjugated secondary antibodies, by Thermo Fisher Scientific (2 mentions)
Lsm700 upright confocal microscope, by Zeiss (2 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (2 mentions)
Anti rab7, by Cell Signaling Technology (2 mentions)
Anti chmp2b, by Abcam (2 mentions)
Anti ha, by Santa Cruz Biotechnology (2 mentions)
Widefield epifluorescent microscope, by Nikon (2 mentions)
Alexa fluor 488 donkey anti rabbit, by Thermo Fisher Scientific (2 mentions)
Anti phospho her3 tyr1289, by Cell Signaling Technology (2 mentions)
Anti met d1c2, by Cell Signaling Technology (2 mentions)
Anti phospho met tyr1234 y1235, by Cell Signaling Technology (2 mentions)
36 protocols using anti eea1
1
Quantification of Gamma-Secretase Complex
2
Immunofluorescence Assay for Organelle Markers
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Immunofluorescence studies were carried out as previously described [24] (link). Briefly, cells grown overnight on 24-well cover glasses (Menzel, 12 mm) were treated with Cy2/Cy3-labeled MOA in FCS-containing medium and fixed in 4% paraformaldehyde (PFA) at 4 °C for 10 min. Cells were washed and incubated with NH4Cl at room temperature for 15 min before being permeabilized with PBS containing 0.02% saponin and 0.2% bovine serum albumin (BSA). Cells were stained with either anti-EEA1 (1:50, BD Transduction Laboratories), anti-Calnexin (1:100, Enzo Life Science), anti-CTR 433 (1:100, kind gift of Michel Bornens, Curie Institut), anti-Giantin (1:100, Abcam), anti-TGN46 (1:100, Sigma Aldrich), anti-Transferrin receptor (TfR, 1:100, Life Technologies), anti-Rab11 (1:100, Cell Signaling), anti-Lamp1 (1:200, BD PharMingen), anti-Rab7 (1:100, Santa Cruz Biotech) or anti-β1 integrin (1:100, R&D Systems) antibodies diluted in permeabilization buffer, followed by either donkey anti-mouse Cy3-labeled secondary antibody (1:100, Jackson ImmunoResearch), donkey anti-rabbit Cy3-labeled secondary antibody (1:100, Jackson ImmunoResearch) or donkey anti-goat Cy3-labeled secondary antibody (1:100, Jackson ImmunoResearch) diluted in permeabilization buffer. Nuclei were stained using DAPI (300 nM, Life Technologies).
3
Comprehensive Antibody Inventory for Cell Organelle Labeling
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Anti-HA antibody (#3F10) was purchased from Roche Diagnostics; anti-CERT (#ab72536) from Abcam; chicken antibody against VAP (Kumagai et al., 2014 (link)) anti-TGOLN2/TGN46 (#A304-434A) from Bethyl Laboratories; anti-TOMM20 (#WH0009804M1) from Sigma-Aldrich; anti-Lamp2 (#sc-18822) from Santa Cruz; anti-LBPA (#z-PLBPA) from Echelon Bioscience; anti-EEA1 (#610457) from BD Transduction Laboratories; anti-catalase (#D4P7B) and anti-Rab11a (#2413) from Cell Signaling Technology; anti-Hrs (#10390-1-AP) from Proteintech; anti-GFP (#04404-84) from Nacalai Tesque, and anti-GAPDH (016-25523) from Fujifilm Wako Pure Chemical Corporation. 2-Bromohexadecanoic acid (2-BP, #M1177) was purchased from Sigma-Aldrich. Lipid dye II (#LD02) was from Dojindo Laboratories. Lysenin was a gift from Dr. Sekizawa (Zenyaku Kogyo). GFP-lysenin was a gift from Dr. Kobayashi (Riken). [14C(U)]L-serine (156 mCi/mmol, #MC265) was from Moravec.
4
Aptamer-Mediated Protein Detection in Cells
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Adherent cells were plated on sterile glass coverslips for one night at 37 °C in culture medium, washed three times, and then saturated for 1 h at room temperature (RT) in selection buffer (phosphate-buffered saline, 1 mM MgCl2, 0.5 mM CaCl2; pH 7.4) containing 2% BSA. Labeled aptamers were denatured at 95 °C for 3 min, incubated on ice for 5 min before being resuspended in selection buffer, and applied to cells for 30 min at 37 °C. Cells were then washed in selection buffer, fixed for 8 min in 4% paraformaldehyde (PFA), permeabilized for 2 min with 0.2% Triton, and washed again. Then, immunocytochemistry was performed with the following primary antibodies: anti-EGFR (clone D1D4J; Cell Signaling Technology; 1/200) and anti-EEA1 (early endosome antigen 1; clone 14/EEA1; BD Transduction Laboratories; 1/1000). Primary antibodies were added overnight (O/N) at 4 °C, followed by two washes and incubation for 1 h at RT with a secondary antibody conjugated to Alexa 488 or 568 (Life Technologies, Carlsbad, CA, USA) at a 1 µg/mL final concentration. DAPI was added at 1 µg/mL to visualize nuclei. Washing steps were performed before mounting using fluorescent mounting medium (S3023; Dako, Carpinteria, CA, USA).
5
Evaluating EGFR Signaling Dynamics
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Gefitinib (AstraZeneca), Phenylarsine Oxide (PAO) (Sigma‐Aldrich), Filipin III (Sigma‐Aldrich), anti‐EGFR (sc‐373746), anti‐EGFR (4267, Cell signaling), anti‐p‐EGFR (2234, Cell signaling), anti‐STAT3 (sc‐8019), anti‐p‐STAT3 (sc‐8059), anti‐ERK (9102, Cell signaling), anti‐p‐ERK (9101, Cell signaling), anti‐EEA1 (610457, BD Biosciences), anti‐PARP (9542, Cell signaling), anti‐c‐Myc (9402, Cell signaling), anti‐β‐actin (A5316), Goat anti‐mouse IgG (H + L)‐HRP conjugate (1706516, Bio‐Rad), Goat anti‐rabbit IgG polyclonal HRP conjugated (ADI‐SAB‐300, Enzo), Alexa Fluor 488 goat anti‐rabbit antibody (A32731, Thermo Fisher Scientific), and Alexa Fluor 594 goat anti‐mouse antibody (A32742, Thermo Fisher Scientific).
6
Immunocytochemistry of Hippocampal Neurons
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Hippocampal cell culture was fixed with 4% PFA/4% sucrose/PBS, pH7, for 5min. The cells were permeabilized with block solution (PBS, 10% horse serum, and 0.5% BSA, 0.2% Triton) for 10min. The cells were incubated with primary antibodies including, anti-GLT1 (1:500, Alomone), anti-EEA1 (1:500, BD Bioscience, Cat. No: 610456), anti-Map2 (1:1000) for 1hr in block solution, followed by incubation with Alexa Fluor-488 or 555 or 647-conjugated secondary antibodies (1:1000, Invitrogen) for 1hr. After extensive washing, coverslips were mounted on microscope slides using ProLong Gold antifade reagent (Invitrogen) and sealed with nail varnish. Images were attained using Zeiss LSM 700 upright confocal microscope with an Apochromat 63× oil immersion lens with 1.4 numerical aperture. Images were digitally captured using ZEN software with excitation at 488nm for GFP and Alexa-Fluor 488, 555nm
for Alexa-Fluor 555 and 633nm for Alexa-Fluor 647 and conjugated secondary antibodies.
Pinholes were set to 1 Airy unit creating an optical slice of 0.8μm. For measuring colocalization, the colocalization plugin of the Metamorph software was used. Fluorescence intensity was measured using the ImageJ software.
for Alexa-Fluor 555 and 633nm for Alexa-Fluor 647 and conjugated secondary antibodies.
Pinholes were set to 1 Airy unit creating an optical slice of 0.8μm. For measuring colocalization, the colocalization plugin of the Metamorph software was used. Fluorescence intensity was measured using the ImageJ software.
7
Antibody Characterization for Vesicle Trafficking
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TAT1 anti-tubulin was a gift from Keith Gull (University of Oxford). The following commercial antibodies were used. Mouse: anti-LITAF (Santa Cruz); anti-transferrin receptor (Zymed); anti-Strep-Tag (Novagen); anti-CD63 (Millipore); anti-EEA1 (early endosome antigen 1; BD Biosciences); anti-LAMP1 (lysosome-associated membrane protein 1; Developmental Studies Hybridoma Bank, University of Iowa); anti-OPG2 (opsin tag containing two glycosylation sites) was described previously [29 (link)]. Rabbit: anti-TorsinA was a gift from Lisa Swanton (University of Manchester); anti-V5 (Abcam); anti-EEA1, anti-LAMP1 (Cell Signaling). Sheep: anti-GFP was an in-house antibody generated against GST-GFP. Fluorescent secondary antibodies for IF and for Licor immunoblotting were from Jackson ImmunoResearch Laboratories (PA, USA).
8
Immunofluorescence Staining of Mitochondria and Endosomes
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Cells were fixed for 15 min with 4% paraformaldehyde (PFA) at 37°C, permeabilized with 0.2% TritonX-100 in PBS for 15 min at room temperature, blocked for 90 min on a gentle rocker-shaker in blocking buffer [0.5% fish skin gelatin (FSG)/1x PBS/1% bovine serum albumin (BSA), 0.2% Triton X-100).14 (link),15 (link),25 (link),66 (link) Cells were immunostained with a monoclonal primary antibody anti-Tom20 (Santa Cruz Biotechnology, Inc sc-11415) and anti-EEA1 (BD Biosciences, 610456), then incubated with Alexa Fluor secondary antibodies for 1 h, post-fixed with 4% PFA for 10 min, and stained with DAPI for 15 min. All solutions were 0.45 μm syringe filtered.
9
Detailed Immunofluorescence Protocol
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All phospholipids were obtained from Avanti Polar Lipids. Primary antibodies were obtained from the following companies: anti-GM130, anti-EEA1, and anti-p150Glued from BD Biosciences; anti-LAMP1, anti–α-tubulin, and anti-mitofilin from Abcam; anti-FLAG from Sigma-Aldrich; anti-dynein intermediate chain from Millipore; anti-PDI from ABfinity; anti-GFP, anti-syntaxin 6, anti-syntaxin 13, anti-vti1a, anti-VAMP4, and anti-Rab5 from Synaptic Systems; anti-LC3 from MBL International. Cy2- or Cy3-conjugated goat anti-mouse and goat anti-rabbit IgG and HRP-conjugated goat anti-mouse and goat anti-rabbit IgG and rabbit anti-sheep IgG were obtained from Jackson ImmunoResarch Laboratories. Alexa Fluor-conjugated transferrins, streptavidin-conjugated β-galactosidase, and 5-dodecanoylaminofluorescein di-β-d -galactopyranoside (C12FDG) were from Molecular Probes. The 100-µm fluorescent polystyrene beads, biotin-conjugated transferrin, calcein, N-ethylmaleimide, and DAPI were from Sigma-Aldrich. Ciliobrevin D was from Millipore.
10
Immunofluorescence Staining of Subcellular Structures
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The cells were washed with the phosphate buffered saline (PBS), fixed in 2–4% formaldehyde (prepared from paraformaldehyde) in PBS for 5–15 min at room temperature (RT) and permeabilized with Triton X-100 for 10 min at RT. The non-specific background staining was reduced by incubating cells in blocking buffer, containing 3% bovine serum albumin (BSA) and 10% goat serum in PBS, at 37°C for 1 h. The cells were then stained with primary antibodies, diluted into 3% BSA in PBS and incubated at 37°C for 2 h or at 4°C overnight. When two primary antibodies (raised in different species) were used, the staining was done sequentially. Afterwards, the cells were rinsed in PBS and stained with secondary antibodies at 37°C for 45 min. At the end of the staining protocol the cells were mounted onto glass slides using Slowfade Gold antifade reagent (Molecular Probes, Invitrogen).
Primary antibodies used were: anti-E 1∶10 [35] (link), anti-β-Actin 1∶200 (Abcam), anti-α-Tubulin 1∶100 (Sigma), anti-Clathrin light chain 1∶300 (Synaptic Systems), anti-EEA1 1:300 (BD Biosciences) and anti-LAMP1 1:300 (Abcam). Secondary antibodies were Alexa Fluor® 488 goat anti-mouse IgG and Alexa Fluor® 546 goat anti-rabbit IgG (Molecular Probes, Invitrogen).
Primary antibodies used were: anti-E 1∶10 [35] (link), anti-β-Actin 1∶200 (Abcam), anti-α-Tubulin 1∶100 (Sigma), anti-Clathrin light chain 1∶300 (Synaptic Systems), anti-EEA1 1:300 (BD Biosciences) and anti-LAMP1 1:300 (Abcam). Secondary antibodies were Alexa Fluor® 488 goat anti-mouse IgG and Alexa Fluor® 546 goat anti-rabbit IgG (Molecular Probes, Invitrogen).
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