All reagents were from Sigma, except where indicated, with the following stock compositions. Yeast nitrogen base (Difco); Choline (1 M stock in DDW); Rapamycin (GoldBio; primary stock solution: 2.5 mg/ml in 100% ethanol, and secondary stock of 1 mg/ml in 90% ethanol+10% Tween‐20 (v:v), final 200 ng/ml). Copper sulfate (used at 500 μM from a 0.5 M stock in DDW). Potassium Permanganate (Dissolved in DDW), S‐1 (or Dimethylethanolamine)—Ted‐Pella−18315. BEEM embedding capsules size 00, EMS, 70010‐B. Proteinase K (stock 2 mg/ml, final 100 mg/ml (diluted in PS200)); Triton X‐100 (stock 40% (w/v), final 0.4% (w/v)); DTT (Bio Basic Inc); PMSF (200 mM in EtOH); Z1000 Zymolyase 20T (US biological); Sodium‐Sulfate (BDH); Mouse anti‐GFP (MBL); mouse anti‐Pgk1 (Abcam); Rabbit anti‐Ape1 (polyclonal antibodies prepared by using two synthetic peptides that were conjugated to keyhole limpet hemocyanin (KLH) and then injected into rabbits to produce an anti‐Ape1 antiserum that recognizes both precursor and mature forms); Anti‐cpy1 polyclonal antibody (obtained from Prof. Howard Riezman); pRS316‐GFP‐Atg8 (expressed under the promoter of Atg8, kind gift from Prof. Yoshinori Ohsumi); pDP105 (expressing TagBFP‐Ape1 under the native Ape1 promoter and Ape1 under the pCUP1 promoter, kind gift from Prof. Claudine Kraft).
Mouse anti gfp
Manufactured by Abcam
Sourced in United Kingdom, United States
Mouse anti-GFP is a primary antibody that binds to the green fluorescent protein (GFP). It is used for the detection and quantification of GFP-tagged proteins in various applications, such as Western blotting, immunohistochemistry, and flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Rhodamine phalloidin, by Thermo Fisher Scientific (6 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (4 mentions)
Rabbit anti zo 1, by Thermo Fisher Scientific (4 mentions)
Mouse anti myc, by Santa Cruz Biotechnology (3 mentions)
Rabbit anti ha, by Abcam (3 mentions)
Rabbit anti rfp, by Abcam (2 mentions)
Phalloidin atto 488, by Merck Group (2 mentions)
Alexa fluor 488 donkey anti rabbit, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 647 phalloidin, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 555 goat anti rabbit, by Thermo Fisher Scientific (2 mentions)
Mouse anti ck20, by Agilent Technologies (2 mentions)
Rabbit anti olfm4, by Cell Signaling Technology (2 mentions)
Goat anti mouse alexa fluor 405, by Abcam (2 mentions)
Rat anti e cadherin eccd 2, by Thermo Fisher Scientific (2 mentions)
Rabbit anti lysozyme, by Agilent Technologies (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Mouse anti ki67, by BD (2 mentions)
Mouse anti gst, by Proteintech (2 mentions)
Rabbit anti glun2a, by Merck Group (2 mentions)
Hrp conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
24 protocols using mouse anti gfp
1
Yeast Autophagy Protein Localization
2
Immunofluorescence Microscopy Technique
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Immunofluorescence was performed as previously described (Oegema et al., 2001 (link)), with slides fixed in methanol for 40–45 min. The following antibodies were used: rabbit anti-AIR-2 (1:500; gift from Jill Schumacher), rabbit anti-BUB-1 (1:2800; gift from A Desai), rabbit anti- DHC-1 (1:100, gift from P Gönczy), rabbit anti-GFP (1:500; AbCam, Cambridge, UK), mouse anti-GFP (1:500; AbCam), rabbit anti-KLP-18 (1:10,000, gift of O Bossinger), rabbit anti-KNL-3 (1:3800; gift from A Desai), mouse anti-MPM-2 (1:500; AbCam and Millipore, Billerica, MA), rabbit anti-SEP-1 (1:200; gift from A Golden), mouse anti-α-tubulin-FITC (1:500; Sigma). Alexa-flour-conjugated secondary antibodies (Invitrogen, Carlsbad, CA) were used at 1:500.
3
Immunolocalization of Cytoskeletal Proteins in Plant Roots
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Immunolocalization of microtubules, PLDα1, PLDα1-YFP, and clathrin in root wholemounts was done as described previously (Šamajová et al., 2014 (link)). Samples were immunolabeled with rat anti-α-tubulin (clone YOL1/34; ABD Serotec), rabbit anti-phospholipase D alpha 1/2 (Agrisera, Sweden), mouse monoclonal anti-clathrin LC (Sigma-Aldrich) or mouse anti-GFP (Abcam) primary antibodies diluted 1:300, 1:300, 1:300 and 1:100, respectively, in 3% (w/v) BSA in PBS at 4°C overnight. In the case of double or triple co-immunolocalization a sequential immunolabeling was performed. Secondary antibodies included Alexa-Fluor 488 goat anti-rat, Alexa-Fluor 488 goat anti-mouse or Alexa-Fluor 546 goat anti-rat IgGs were diluted 1:500 in PBS containing 3% (w/v) BSA for 3 h (1.5 h at 37°C and 1.5 h at room temperature). Where necessary, nuclei were counterstained with DAPI. Microscopic analysis of immunolabeled samples was performed with a Zeiss 710 CLSM platform (Carl Zeiss, Jena, Germany), using excitation lines at 405, 488, and 561 nm from argon, HeNe, diode, and diode pumped solid-state lasers.
4
GFP and p85 Immunoprecipitation Protocol
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Cells were washed with PBS prior to lysis in lysis buffer (20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], pH 7.4, 110 mM potassium acetate, 2 mM magnesium chloride, 0.1% [vol/vol] Tween 20, 1% [vol/vol] Triton X-100, 0.5% [wt/vol] sodium deoxycholate, and 500 mM sodium chloride), supplemented with Complete protease inhibitor and PhosSTOP phosphatase inhibitor cocktails (Roche) on ice. After centrifugation (20,000×g for 10 min, 4°C), cell lysates were incubated with mouse-anti-GFP (Abcam, ab1218) or rabbit-anti-p85 (Cell Signaling Technology, catalog numbers 4257 and 4292, used at 1:1 ratio) antibodies for 20 min at room temperature by using a rotator, followed by incubation with protein G magnetic beads (GE Healthcare) at 4°C overnight. Immunoprecipitation samples were washed with lysis buffer, eluted with 4× reducing NuPAGE LDS sample buffer (Life Technologies), heated at 95°C for 5 min and analyzed by SDS-PAGE and western blotting.
5
Western Blot Analysis of Neuronal Proteins
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Western blot assays were followed according to an established protocol32 (link). Briefly, dissected cerebral cortex tissues, cervical spinal cord tissues or cells were lysed in RIPA lysis buffer (Cell Signal Technology, Beverly, MA). Protein concentrations were determined using a BCA assay (Pierce, Roclford, IL, USA) and equal amounts were loaded onto 10% polyacrylamide gel (Bio-Rad, Hercules, CA, USA) and separated by SDS-PAGE. Samples were then transferred to PVDF membranes, blocked in 5% nonfat dried milk (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), and incubated overnight at 4 °C with primary antibodies. Antibody labelling was detected by incubation with horseradish peroxidase-labelled anti-rabbit/mouse secondary antibody (Proteintech, Wuhan, China). Protein was visualized using chemiluminescence (Millipore). Antibodies evaluated by Western blot included rabbit anti-p-PKCγ (Abcam), rabbit anti-GAP43 (Abcam), mouse anti-β-Catenin (Abmart), rabbit anti-phospho-GSK-3β (Ser9) (Cell Signal Technology), mouse anti-GAPDH (Thermo Fisher Scientific), mouse anti-β-actin (Thermo Fisher Scientific), mouse anti-HA-tag (Abmart), and mouse anti-GFP (Abcam).
6
Immunofluorescence Staining of Drosophila Brains
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The brains of ice anesthetized flies were dissected in PBS and then fixed in 4% PFA on ice for at least 1 hr. Fixed brains were incubated in Penetration/Blocking Buffer (2% Triton X-100 and 10% Calf Serum in PBS) for 20–24 hr at 4°C and incubated in Dilution Buffer (0.25% Triton X-100 and 1% Calf Serum in PBS) with primary antibodies at 4°C for 20–24 hr. The samples were then washed in Washing Buffer (3% NaCl and 1% Triton X-100 in PBS) for 3 × 30 min at room temperature and subsequently incubated in secondary antibodies for 20–24 hr at 4°C. The brains were washed again in Washing Buffer before mounted in Fluoroshield with DAPI (Sigma-Aldrich) for confocal imaging (Nikon 60 × A/1.20 WI). Antibodies were used at the following dilutions: mouse anti-nc82 (1:200, DSHB), rabbit anti-GFP (1:500, Life Technologies), mouse anti-GFP (1:500, abcam), rabbit anti-dInR (1:100, Cell Signaling Technology), Alexa Fluor 546 goat anti mouse (1:300, Life Technologies), Alexa Fluor 488 goat anti rabbit (1:300, Life Technologies), Alexa Fluor 546 goat anti rabbit (1:300, Life Technologies), and Alexa Fluor 488 goat anti mouse (1:300, Life Technologies).
7
Diverse Antibodies for Cellular Imaging
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The following primary antibodies were used: rabbit anti-Egl [31 (link)] (kindly provided by R. Lehmann); mouse anti-Dhc (Developmental Studies Hybridoma Bank; donor J. Scholey); mouse anti-BicD (Developmental Studies Hybridoma Bank, donor R. Steward); rat anti-Vasa (Developmental Studies Hybridoma Bank; donors A.C. Spradling/D/Williams); rabbit anti-gamma-tubulin (Sigma); mouse anti-GFP (Abcam); mouse anti-HA (Cell signaling technology); rabbit anti-HA (Cell signaling technology); rabbit anti-HOAP, guinea pig anti-HipHop and anti-Gag HeT-A (kindly provided by Y. Rong); rabbit anti-Gag HeT-A (kindly provided by E. Casacuberta (23) Alexa fluor conjugated secondary antibodies (Life Technologies) were diluted 1:500.
8
Immunofluorescence Labeling of Transfected Cells
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MDCK cells or COS7 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and penicillin (40 U/ml)/streptomycin (40 μg/ml) at 37°C under 5% CO2. Cells were transiently transfected with 1 μg of plasmids of interest using Lipofectamine 2000.
For cell-culture labeling, cells were cultured on poly-L-lysine-coated glass coverslips and transfected. 24 hours after transfection, cells were fixed with 4% PFA in 4% sucrose-PBS for 10 minutes and then permeabilized with 0.25% Triton X-100 in PBS. Detection was performed using mouse anti-GFP (Abcam, 1:500), rabbit anti-Harmonin (SDIX, 1:100) and rabbit anti-Ift88 (Krock and Perkins, 2008 (link)) antibodies. Secondary antibodies were goat anti-mouse Alexa-Fluor-488-conjugated and goat anti-rabbit Alexa-Fluor-568-conjugated (Molecular Probes, 1:250).
For cell-culture labeling, cells were cultured on poly-L-lysine-coated glass coverslips and transfected. 24 hours after transfection, cells were fixed with 4% PFA in 4% sucrose-PBS for 10 minutes and then permeabilized with 0.25% Triton X-100 in PBS. Detection was performed using mouse anti-GFP (Abcam, 1:500), rabbit anti-Harmonin (SDIX, 1:100) and rabbit anti-Ift88 (Krock and Perkins, 2008 (link)) antibodies. Secondary antibodies were goat anti-mouse Alexa-Fluor-488-conjugated and goat anti-rabbit Alexa-Fluor-568-conjugated (Molecular Probes, 1:250).
9
Generation of CFAP53 Antibodies
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cDNA for the C-terminal region of mouse CFAP53 was cloned into the bacterial expression vector pMaL-c2X (New England Biolabs) with the use of the forward and reverse primers 5'-AGAGTCTATGCAGCGGGAGT-3' and 5'-TAGAGGAGGCTTGTCAGGGTA-3', respectively. CFAP53 expressed as a fusion protein with maltose binding protein in Escherichia coli was purified from the bacterial cells with the use of amylose resin (New England Biolabs) and injected into rabbits for the generation of antibodies. Specific antibodies were purified from rabbit serum by affinity chromatography. Additional antibodies used in this study are rabbit anti-GFP (Invitrogen, A-11122), mouse anti-acetylated Tubulin (Sigma, T6793), mouse anti- γ-Tubulin (Sigma, T5326), mouse anti-PCM1 (Santa Cruz, SC-398365), mouse anti-human DNAI2 (Abnova, H00064446-MO1), mouse anti-α-Tubulin (Sigma, T5168), mouse anti-HA (Santa Cruz, SC7392), mouse anti-GFP (Abcam, ab1218), rabbit anti-GFP (Santa Cruz SC-8334) mouse anti-MYC (Santa Cruz, SC-40), rabbit anti-HA (Santa Cruz, SC-805) and rabbit anti-MYC (Santa Cruz, SC-289). Antibodies to DNAH5 and DNAH9 were kindly provided by H. Takeda, described previously [41 (link)]. Rabbit antibodies to TTC25 were described previously [42 (link)]. Alexa Fluor-conjugated secondary antibodies for immunofluorescence staining were obtained from Invitrogen.
10
Immunofluorescence Staining of Mouse Spinal Cord
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Spinal columns were removed after mice were perfused with 4% paraformaldehyde (PFA). For immunofluorescence assay, free floating sections of the lumbar spinal cord were prepared as previously described (Huang et al., 2006 (link)). For immunofluorescence assay, sections were blocked with 10% normal serum for 2 h and stained with primary antibodies at 4°C for 24–48 h. After washing with PBS-T (PBS with 0.1% Triton X-100; Sigma-Aldrich) three times, the sections were incubated with secondary antibodies at room temperature for 2 h and mounted in ProLong Gold antifade reagent (Invitrogen). Antibodies used include rat anti-CD11b (BD), mouse anti-GFP (Abcam), rabbit anti-RFP (Abcam), Alexa Fluor 488 goat anti–mouse IgG (Invitrogen), Alexa Fluor 594 goat anti–rabbit IgG (Invitrogen), and Alexa Fluor 647 goat anti–rat IgG (Invitrogen). Nuclei were labeled by DAPI. Images were collected by confocal laser-scanning microscope (SP5; Leica).
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