Ab1218

Manufactured by Abcam

Sourced in United States, United Kingdom

Ab1218 is a lab equipment product. It is a tool designed for use in research laboratories. The core function of Ab1218 is to facilitate specific laboratory tasks, but a detailed description cannot be provided while maintaining an unbiased and factual approach.

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Lab products found in correlation

Ab290, by Abcam (9 mentions) Triton x 100, by Merck Group (4 mentions) Alexa fluor 488, by Thermo Fisher Scientific (4 mentions) Ab6556, by Abcam (4 mentions) Polyvinylidene difluoride membrane, by Merck Group (3 mentions) Alexa fluor 568, by Thermo Fisher Scientific (3 mentions) Phosphate buffered saline (pbs), by Thermo Fisher Scientific (3 mentions) Bovine serum albumin (bsa), by Merck Group (3 mentions) A11001, by Thermo Fisher Scientific (3 mentions) Ab6728, by Abcam (2 mentions) Molecular weight protein standards, by Bio-Rad (2 mentions) Alexa fluor 488 4412, by Cell Signaling Technology (2 mentions) Gapdh antibody g9545, by Merck Group (2 mentions) Nucleoporin c39a3, by Cell Signaling Technology (2 mentions) Runx2 mouse monoclonal antibody sc 390351, by Santa Cruz Biotechnology (2 mentions) Cd44 icd antibody kal ko601, by Cosmo Bio (2 mentions) Horseradish peroxidaseconjugated anti rabbit and anti mouse secondary antibodies, by LGC (2 mentions) Polyacrylamide gel electrophoresis page reagents, by Bio-Rad (2 mentions) Prolong gold antifade dapi 8961, by Cell Signaling Technology (2 mentions) Protein assay reagent, by Bio-Rad (2 mentions)

Close spelling variants of this product

Mouse anti-GFP (ab1218) (2 citations)

66 protocols using ab1218

Investigating ICP27 Interactions in HSV-1 Infection

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HeLa cells were grown on minimal essential medium containing 10% newborn calf serum. HSV-1 wild type strain KOS, null mutant 27-LacZ, N-YFP-tagged ICP27 (N-YFP-ICP27) and n504 were previously described31 (link). Cells were infected with wild-type or mutant virus as indicated for 8 hours at a multiplicity of infection (MOI) of 10 for single infections and a MOI of 5 for co-infections and incubated at 37 °C. Transfection of plasmid DNA was performed by using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s protocol. Transfected cells were infected 24 hours after transfection with 27-LacZ to stimulate expression of the native ICP27 promoter by the virion tegument protein VP16 as previously described58 (link). Eight hours after infection, cells were harvested, and immunoprecipitation was performed on cell lysates using GFP/YFP antibody Ab290 (Abcam) as described previously6 (link). Immunoprecipitated complexes were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose. Western blot analysis was performed as described previously6 (link) with anti-ICP27 antibodies P1119 and P1113 (Virusys), anti-GFP/YFP (Ab290; Abcam), anti-GFP antibody Ab1218 (Abcam) and Lamin B1 (Abcam).

Tunnicliffe R.B., Schacht M., Levy C., Jowitt T.A., Sandri-Goldin R.M, & Golovanov A.P. (2015). The structure of the folded domain from the signature multifunctional protein ICP27 from herpes simplex virus-1 reveals an intertwined dimer. Scientific Reports, 5, 11234.

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Quantitative Protein Analysis by Western Blot

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Cells were washed with cold PBS, and lysed in cold lysis buffer (150 mM NaCl, 50mM Tris-Cl, pH 7.4, 1 mM EDTA, 1% Triton, complete mini tablet-protease inhibitor cocktail tablets [Roche]). The proteins were separated by 10% SDS–PAGE and transferred to PVDF membranes. After blocking with 5% BSA in TBS with Tween (TBST), the membrane was incubated overnight at 4°C with one of the following primary antibodies: GADPH (CST, 5174S), flag (9A3, Mouse monoclonal, CST, 8146T), ADAM17 (Abcam, ab13535), and GFP (Abcam, ab1218). Next day, the membrane was washed with TBST for three times, and incubated with corresponding anti-mouse/rabbit/goat secondary antibodies (mouse, Abcam, ab6728) (rabbit, Abcam, ab6721) (goat, Abcam, ab6789) at 1:10000 dilution at room temperature for 1 h. Signal intensity was quantified with ImageJ software.

Xie Y., Ma A., Wang B., Peng R., Jing Y., Wang D., Finnell R.H., Qiao B., Wang Y., Wang H, & Zheng Y. (2019). Rare mutations of ADAM17 from TOFs induce hypertrophy in human embryonic stem cell-derived cardiomyocytes via HB-EGF signaling. Clinical Science (London, England : 1979), 133(2), 225-238.

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Cloning and Tagging of Murine and Human Lipid Regulators

Cited 1 time

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The coding region of murine Cidea, Cideb, Cidec, Dff40, and Dff45 were cloned into the vector pcDNA3.1D/V5-His-TOPO (Invitrogen, Paisley, UK) to obtain the v5-tagged versions of the proteins (Hallberg et al., 2008 (link)). The human full-length and truncated forms of Cidec were subcloned into pcDNA3.1D/V5-His-TOPO from their GFP constructs (Rubio-Cabezas et al., 2009 (link)) and Lipin-1γ-v5 was constructed from pGH321 (Han and Carman, 2010 (link)). Mutations and deletions were generated with the QuikChange Lightning Kit (Agilent). Tagged proteins were detected by using antibodies against v5 (R96025; Invitrogen), HA (H6908; Sigma), or GFP (ab1218; Abcam).

Barneda D., Planas-Iglesias J., Gaspar M.L., Mohammadyani D., Prasannan S., Dormann D., Han G.S., Jesch S.A., Carman G.M., Kagan V., Parker M.G., Ktistakis N.T., Klein-Seetharaman J., Dixon A.M., Henry S.A, & Christian M. (2015). The brown adipocyte protein CIDEA promotes lipid droplet fusion via a phosphatidic acid-binding amphipathic helix. eLife, 4, e07485.

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Immunofluorescence Staining for Lung Cell Types

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Paraffin sections were de-waxed and rehydrated. Antigen retrieval was carried out by microwaving the slides in 0.01 M sodium citric acid buffer (pH 6.0) for 30 min. Sections were then immersed for 1 hour in blocking buffer (3% BSA, 0.2% Triton X-100 in PBS), then incubated in primary antibody (in blocking buffer) at 4° C overnight, followed by incubation with secondary antibody at 4° C for 1 hour. Slides were mounted with antifade reagent with or without DAPI (Life Technologies), and then scanned with a high-resolution MIRAX MIDI system (Carl Zeiss) equipped with both bright field and fluorescence illumination. Images were analyzed by the MIRAX Viewer software.
Polyclonal rabbit anti-Scgb1a1 antibody (US Biological, C5828) was used at 1:200 dilution. Goat anti-pro-SPC (Santa Cruz Biotechnology, sc-7706), rabbit anti-Cyp2f2 (Santa Cruz Biotechnology, sc-67283), goat anti-PDPN (R&D Systems, AF3244), monoclonal mouse anti-p63 4A4 (Santa Cruz Biotechnology, sc-8431), rabbit anti-GFP (Abcam, ab290), goat anti-GFP(Abcam, ab5450), and mouse anti-GFP (Abcam, ab1218) were used at 1:50 dilution. Secondary antibodies (including donkey anti-rabbit, anti-goat, or anti-mouse) each with different Alexa Fluor conjugations were all purchased from Life Technologies, and used at 1:200 dilution.

Zheng D., Soh B.S., Yin L., Hu G., Chen Q., Choi H., Han J., Chow V.T, & Chen J. (2017). Differentiation of Club Cells to Alveolar Epithelial Cells In Vitro. Scientific Reports, 7, 41661.

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Antibody Validation and Detection Protocol

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For this study, we used antibodies to MTSS1 (1:30 for IHC, LS-B1818, LifeSpan BioSciences, Seattle, WA, USA), MTSS1 (1:250 for IB, 4386, Cell Signaling Technology, Danvers, MA, USA), MTSS1 (1:50 for PLA, sc-101390, Santa Cruz Biotechnology, Dallas, TX, USA), SCAMP1 (1:25 for IHC, 1:100 for PLA, 15327-1-AP, Proteintech, Rosemont, IL, USA), SERPIN B13 (1:125 for IHC, NBP2-01312, Novus Biological, Littleton, CO 80120, USA), GFP (1:1000, ab1218, Abcam, Cambridge, UK), HA (1:1000 for IB, 1:200 for IF, A190-108 A, Bethyl Laboratories Inc., Montgomery, TX, USA), β-actin (1:5000 for IB, A5441, Sigma-Aldrich, St Louis, MO, USA).

Vadakekolathu J., Al-Juboori S.I., Johnson C., Schneider A., Buczek M.E., Di Biase A., Pockley A.G., Ball G.R., Powe D.G, & Regad T. (2018). MTSS1 and SCAMP1 cooperate to prevent invasion in breast cancer. Cell Death & Disease, 9(3), 344.

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Immunoblotting of Fluorescent Fusion Proteins

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HEK293T cells were resuspended in lysis buffer and sonicated. After centrifugation (15,000 x g, 4 °C, 5 min), the protein concentration was determined in the lysate by a Lowry protein assay kit (Bio-Rad Laboratories GmbH, Munich, Germany). For immune detection of Clover, mRuby2, Clover fused (63bp) mRuby2, Clover-PPARγ1, mRuby2-RXRα, mRuby2-RXRα Δ414-462 and N-CoR2-mRuby2 constructs, 100 µg protein per sample was separated on 10% sodium dodecyl sulfate (SDS) polyacrylamide gels followed by transfer onto nitrocellulose membranes (both Bio-Rad Laboratories GmbH) basically following standard procedures. Subsequently, membranes were incubated with first antibodies against GFP (1:1,000; ab1218, Abcam plc, Cambridge, UK), human PPARγ (1:1,000; D69, Cell Signaling Technology, Danvers, USA), human RXRα (1:1,000; NB100-1466, Novus Biologicals, LLC, Littleton, USA), human N-CoR2 (1:1,000; ab2781, Abcam plc) and tRFP (1:1,000; AB233, Evrogen Joint Stock Company, Moscow, Russia) followed by Alexa Fluor^® 488, 546 or 647 (Life Technologies Inc., Carlsbad, USA) fluorescent dyes secondary antibodies (1:10,000). Blots were visualized using the ChemiDoc XRS+ system (Bio-Rad Laboratories GmbH).

Trümper V., von Knethen A., Preuß A., Ermilov E., Hackbarth S., Kuchler L., Gunne S., Schäfer A., Bornhütter T., Vereb G., Ujlaky-Nagy L., Brüne B., Röder B., Schindler M., Parnham M.J, & Knape T. (2019). Flow cytometry-based FRET identifies binding intensities in PPARγ1 protein-protein interactions in living cells. Theranostics, 9(19), 5444-5463.

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Antibody Source and Characterization

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We obtained antibodies to CD44 [156–3C11], RUNX2 [D1L7F], SOX2 [D6D9], MMP-9 [D6O3H], green fluorescent protein (GFP) [D5.1], and nucleoporin [C39A3] from Cell Signaling Technology, Inc. (Danvers, MA, USA). RUNX2 mouse monoclonal antibody (sc-390351) was purchased from Santa Cruz Biotechnology, Inc. CD44-ICD antibody (KAL-KO601) was purchased from Cosmo Bio. Antibodies against CD44 (ab157107) and GFP (ab1218) were purchased from Abcam. Chemicals and GAPDH antibody (G9545) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Horseradish peroxidaseconjugated anti-rabbit and anti-mouse secondary antibodies were obtained from Kirkegaard & Perry Laboratories (Gaithersburg, MD, USA) and Santa Cruz Biotechnology, respectively. Protein assay reagents, molecular weight protein standards, and polyacrylamide gel electrophoresis (PAGE) reagents were purchased from Bio-Rad (Hercules, CA, USA). Polyvinylidene difluoride membranes were obtained from Millipore Corp. (Bedford, MA, USA). Enhanced chemiluminescence reagent was purchased from Pierce (Rockford, IL, USA). Fluorochrome-conjugated secondary antibodies Alexa Fluor 488 (4412) and ProLong Gold Antifade DAPI (8961) were obtained from Cell Signaling Technology, Inc.

Senbanjo L.T., AlJohani H., AlQranei M., Majumdar S., Ma T, & Chellaiah M.A. (2020). Identification of sequence-specific interactions of the CD44-intracellular domain with RUNX2 in the transcription of matrix metalloprotease-9 in human prostate cancer cells. Cancer Drug Resistance, 3(3), 586-602.

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Immunostaining of Drosophila Embryos

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Dechorionated embryos were fixed and stained as per Roddie et al.³¹. Antibodies were diluted in PATx (0.1% Triton-X100 (Sigma-Aldrich), 1% BSA (Sigma-Aldrich) in PBS (Oxoid, Thermo Fisher, MA, USA)). Rabbit anti-GFP (ab290 1:1000; Abcam, Cambridge, UK) or mouse anti-GFP (ab1218 1:200; Abcam) were used to detect GFP-labelled macrophages. Rabbit anti-cDCP-1 (9578S 1:1000; Cell Signaling Technologies), mouse anti-Repo (concentrate of clone 8D12 used at 1:1000; Developmental Studies Hybridoma Bank, University of Iowa, USA) or mouse anti-Futch (supernatant of clone 22C10 used at 1:200; Developmental Studies Hybridoma Bank) were also used as primary antibodies. Goat anti-mouse or goat anti-rabbit secondary antibodies conjugated to AlexaFluor568, AlexaFluor488 (A11036 and A11034; Invitrogen, Thermo Fisher) or FITC (115-095-146; Jackson Immunoresearch, Cambridge, UK) were used to detect primary antibodies; these were diluted from stock solutions made according to the recommendations of the supplier (1:400 in PATx). Stained embryos were stored in DABCO mountant (Sigma-Aldrich) and mounted on slides for imaging.

Armitage E.L., Roddie H.G, & Evans I.R. (2020). Overexposure to apoptosis via disrupted glial specification perturbs Drosophila macrophage function and reveals roles of the CNS during injury. Cell Death & Disease, 11(8), 627.

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Immunohistochemical Analysis of mTOR Pathway

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The following commercial primary antibodies were used: mTOR (CST#2983; 1/100), LAMP2 mouse monoclonal (Abcam, ab25631: 1/100), TGN46 sheep polyclonal (Novus Biologicals NB110-40767: 1/200), Rab1a rabbit monoclonal (CST#13075: 1/100), GFP mouse monoclonal (Abcam ab1218: 1/100), GM130 mouse polyclonal (Novus Biologicals H00002801-B01P: 1/25), Raptor rabbit polyclonal (Merck Millipore, 09-217: 1/100) and secondary antibodies raised in donkey (Jackson ImmunoResearch: 1/500). Cells were processed and imaged as described in (13 (link), 40 (link)). Proximity ligation assays were performed using the Duolink^®In Situ Orange Starter Mouse/Rabbit kit (DUO92102, Sigma) according to manufacturer’s instructions.
For patient samples, slides were de-waxed and rehydrated with antigen retrieval performed using citrate buffer pH 6.0 (Sigma-Aldrich) and a pressurised De-cloaking Chamber (Biocare Medical). The intensity of cytoplasmic staining was assessed by a pathologist (CS) on a semi-quantitative scale from 0-3. High levels of expression in tumour sections were scored three, lower levels of expression scored zero, one or two.
Immunohistochemistry was carried out as previously described (41 (link)). Paraffin-embedded tissue blocks from formalin-fixed tumour samples were sectioned, de-waxed, and rehydrated using standard techniques and 4 μm sections.

Fan S.J., Snell C., Turley H., Li J.L., McCormick R., Perera S.M., Heublein S., Kazi S., Azad A., Wilson C., Harris A.L, & Goberdhan D.C. (2015). PAT4 levels control amino-acid sensitivity of rapamycin-resistant mTORC1 from the Golgi and affect clinical outcome in colorectal cancer. Oncogene, 35(23), 3004-3015.

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Western Blot Analysis of Protein Expression

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The 293 cells were collected and lysed in 1× loading buffer (9156, Takara). After sonication, lysates were centrifuged at 14 000 × g for 30 min. Total proteins were resolved with 7.5% TGX polyacrylamide gels (1610171, Bio-Rad) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore) by reverse electrophoresis. After being blocked, membranes were stained with anti-GFP (1:2000 dilution; ab1218, Abcam), anti-lamin A/C (1:10000 dilution; A19524, ABclonal) or anti-GAPDH (1:2000 dilution; 2118, Cell Signaling Technology) antibodies. Chemiluminescence signals were collected using Amersham Imager 800 (GE).

Gao K., Zhang X., Zhang Z., Wu X., Guo Y., Fu P., Sun A., Peng J., Zheng J., Yu P., Wang T., Ye Q., Jiang J., Wang H., Lin C.P, & Gao G. (2022). Transcription-coupled donor DNA expression increases homologous recombination for efficient genome editing. Nucleic Acids Research, 50(19), e109.

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