HEK293 cells were cultured in 75 cm2 culture flasks and transfected with 10 µg of the appropriate CFP- and HA-tagged constructs in a 1∶1 ratio using the Lipofectamine reagent according to the manufacturer's instructions (Invitrogen). Cells were solubilized 48 hours post-transfection in a 1 x PBS buffer supplemented with 5 mM EDTA, 1% Triton X-100 and a complete protease inhibitor mixture (Roche Diagnostics). Precipitation of the protein complexes from the soluble cell fraction was performed with GFP antibodies (Abcam, Cambridge, UK) and Protein G Agarose beads (Roche Diagnostics) that were pre-blocked with 2% non-fat milk powder in PBS. The proteins were eluted by incubating the beads in NuPAGE LDS sample buffer (Invitrogen) for 15 min at 37 °C. Subsequently, the eluted protein complexes were separated on a 4–12% Bis-Tris SDS-PAGE gel (Invitrogen) and transferred to a polyvinylidine difluoride (PVDF) membrane (GE Healthcare, Buckinghamshire, UK). The blot was blocked with 5% non-fat milk powder in PBS. Immunoprecipitated proteins were detected by incubation of the blots with anti-HA IgG (Roche Diagnostics) followed by incubation with anti-rat IgG conjugated to horseradish peroxidase (GE Healthcare) and subsequent ECL detection (GE Healthcare).
Gfp antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
The GFP antibody is a laboratory reagent used to detect and visualize the presence of green fluorescent protein (GFP) in biological samples. It is a highly specific and sensitive tool for researchers studying gene expression, protein localization, and a variety of other applications involving GFP-tagged proteins.
Automatically generated - may contain errors
Lab products found in correlation
Pms2 gfp, by Addgene (4 mentions)
Magna rip rna binding protein immunoprecipitation kit, by Merck Group (4 mentions)
Flag antibody, by Merck Group (2 mentions)
Th antibody, by Merck Group (2 mentions)
Dapi mounting medium, by Vector Laboratories (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Ecl detection, by GE Healthcare (1 mentions)
Bis tris sds page gel, by Thermo Fisher Scientific (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Lipofectamine reagent, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor mixture, by Roche (1 mentions)
Protein g agarose beads, by Roche (1 mentions)
Enhanced chemiluminescence, by GE Healthcare (1 mentions)
Protease inhibitor mixture, by Merck Group (1 mentions)
Ha antibody, by Santa Cruz Biotechnology (1 mentions)
Mini protean tris glycine gels, by Bio-Rad (1 mentions)
Flag beads, by Takara Bio (1 mentions)
48 protocols using gfp antibody
1
Immunoprecipitation and Western Blot Analysis
2
Protein-Protein Interaction Assay
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HEK293T cells were co-transfected with Flag-tagged target protein, HA-tagged source protein, and GFP-tagged peptide or GFP. Cells were lysed 48 h after transfections with radioimmune precipitation assay buffer (50 mM Tris-HCl pH 7.4, 1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, 10 mM Na3VO4, 10 mM sodium pyrophosphate, 25 mM NaF, 1× protease inhibitor mixture (Sigma)) for 30 min at 4 °C and co-immunoprecipitated with Flag beads (Clontech). The resulting immunocomplexes were analyzed by immunoblot using the appropriate antibodies. Protein samples were separated using 4–20% Mini-PROTEAN Tris-glycine gels (Bio-Rad) transferred to PVDF membranes and blocked in 5% milk containing PBS-Tween-20 (0.1%) for 1 h. PVDF membranes were then incubated with specified primary antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibodies (anti-mouse HRP-linked, Cell Signaling, catalog #7076, 1:5000; anti-rabbit HRP-linked, Cell Signaling, catalog #7074, 1:5000) and detected using enhanced chemiluminescence (GE Healthcare). HA antibodies were obtained from Santa Cruz (catalog #7392, 1:2000), GFP antibodies were purchased from Abcam (catalog #ab290, 1:2000), and Flag antibodies were purchased from Sigma (catalog #A8592, 1:1000).
3
Tracing Engrafted GFP+ Cells via Immunofluorescence
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Immunofluorescence staining of GFP and α‐SMA was carried out for tracing the biological behavior of engrafted GFP+‐AAMSCs in vivo. Briefly, after dewaxing, rehydration, antigen retrieval and blocking, the sections were incubated with mouse anti α‐SMA and GFP antibodies (1:50, Abcam), respectively, overnight at 4°C. Sections were then incubated with the goat anti‐mouse Alexa‐594 conjugated secondary antibody (1:500, Jackson Laboratory) and the goat anti‐chicken Alexa‐488 conjugated secondary antibody (1:500, Jackson Laboratory) for 1 hour. Finally, the nuclei were counterstained with DAPI (1:10,000, Beyotime, Shanghai, China, http://www.beyotime.com/index.html ).
4
Immunohistochemistry and Colony Formation Assay
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Primary antibodies used in immunohistochemistry and immunofluorescence include Phospho-histone H3 (PH3) antibodies (Santa Cruz Biotechnology, sc-8656-R) at 1:100, Yap1 antibodies (Cell Signaling Technology, 4912s) at 1:50, p-Yap1 antibodies (Cell Signal, 4911s) at 1:50, Sox9 antibodies (Abcam, ab3697) at 1:50, Col2a1 antibodies (Santa Cruz, sc-28887) at 1:100, Mmp13 antibodies (Abcam, ab39012) at 1:50, Col10a1 antibodies (Abcam, ab58632) at 1:50 and Runx2 antibodies (Cell Signal, 8486S) at 1:50. The immunohistochemistry was performed using Histostain-Plus IHC kit (Invitrogen) following the manufacturer's protocol. For western blot analysis, b-Actin antibodies (Santa Cruz, sc-130656) at 1:2,000 were used for normalization, Yap1 antibodies (Cell Signal, 4912s) at 1:1000, p-Yap1 antibodies (Cell Signal, 4911s) at 1:1,000, Runx2 antibodies (Cell Signal, 8486S) at 1:1000, Taz antibodies (BD Pharmingen, 560235) at 1:1,000, and GFP antibodies (Abcam, ab290) at 1:2,000 were used. Procedures were followed as previously described (Topol et al., 2003) . Anti-Flag M2 affinity gel A2220) was used for immunoprecipitation.
Colony Formation Assay 5,000 cells were seeded in 60-mm culture dish and cultured for 5 days. Crystal violet staining was performed for counting the colony number. Triplicate plates were counted.
Colony Formation Assay 5,000 cells were seeded in 60-mm culture dish and cultured for 5 days. Crystal violet staining was performed for counting the colony number. Triplicate plates were counted.
5
Purification and Interaction of Chimeric eIF4E1
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To generate the chimeric eIF4E1 protein, we cloned the full-length cDNA of eIF4E1 to the pGEX-4T-1 vector, which contains a GST tag before the multiple cloning sites (MCS). Next, the fused GST-eIF4E1 protein was expressed in E. coli and purified on glutathione affinity resin. The purified fusion protein GST-eIF4E1 was mixed with YFP-tagged RopGEF7 protein (extracted from 35Spro:YFP-RopGEF7 transgenic plants) and then incubated at 4°C for 2 h. The amino acid sequences of GFP and YFP differ only one amino acid, and GFP antibody (Abcam) was used as a substitute for the YFP antibody to detect the YFP-RopGEF7 plant protein. The primers used for plasmid construction are listed in Supplementary Table S1 .
6
Immunofluorescent Localization of GFP
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Sections of 10 µm thickness were rinsed with DPBS for 3 times, blocked with 1% BSA and 0.3% triton for 1 hour, and incubated with GFP antibody (Abcam, MA, USA) overnight at 4 °C. After washing with DPBS for 3 times, Alexa Fluor 546 conjugated donkey anti-rabbit IgG (Thermo Scientific, MA, USA) and 0.1 µg/ml DAPI were used to stained nuclei for 1 hour at room temperature. Slides were sealed by using AQUA-Mount (Thermo Scientific, MA, USA) and dried at room temperature for 2 hours. Photomicrographs were taken on a fluorescence microscope (Olympus, PA, USA).
7
Identification of RNA Binding Proteins
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pMS2-GFP (Addgene) was transfected into stably overexpress CCL14-AS cells. After 48 h, cells were used to perform RIP experiments using a GFP antibody (5 µg per reaction, Abcam, ab290). LoVo cells were lysed in lysis Buffer with RNase inhibitor (Beyotime, 1:1000), DMSF (1:100) and Protease inhibitor (Beyotime,1:100). The protein A/G sepharose beads were washed by lysis Buffer, and the cell extracts were incubated with these beads bound to GFP antibody or control IgG at 4 °C for 2 h. The beads were washed and divided two parts. One part was incubated with proteinase K to remove protein and performed RNA isolation. Other part was used for western blot. Finally, qRT-PCR analysis was performed on the purified RNA, and western blot analysis for Anti-IgG and anti-GFP are performed to detect the quality of the experiment.
8
Primary Neuron Transfection and Treatment
9
RNA-Binding Protein Immunoprecipitation Assay
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pcDNA3.1‐linc00324 and pMS2‐GFP (Addgene plasmids) were cotransfected into CD4+ T cells, and RIP was performed by using GFP antibody (Abcam) and the Magna RIP™ RNA‐Binding Protein Immunoprecipitation Kit (Millipore). Briefly, cells were lysed with radio‐immunoprecipitation assay buffer, and the cell lysates were incubated with antibody‐conjugated magnetic beads suspended in RIP buffer. To isolate the immunoprecipitated RNA, the samples were further digested with proteinase K, followed by purification of RNA for subsequent Illumina sequencing to seek potential target miRNA. Similarly, the anti‐AGO2 antibody (Millipore) was also used in RIP experiment, and the purified RNA was analyzed with qPCR to validate the presence of the binding partners.8
10
ChIP-qPCR analysis of FAMA and MED8
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ProFAMA: FAMA-GFP, ProMED8: MED8-GFP, ProFAMA: FAMA-GFP/med8, ProMED8: MED8-GFP/fama-2 or wild type seedlings were grown in MS medium. For B. cinerea treatment, 5-d-old seedlings of the above materials were sprayed with 0.5×105 to 1.0×105 spores/mL of B. cinerea over a period of 36 h, after which 1.5 g of inoculated entire plants were collected. Additionally, 1.5 g of uninoculated seedlings were collected as a control. The collected seedlings were cross-linked in 1% formaldehyde, and their chromatin isolated [44 ]. A GFP antibody (Abcam, Cambridge, UK) was used to immunoprecipitate the protein-DNA complex, and the precipitated DNA was purified using a PCR purification kit (Qiagen, Hilden, Germany) in preparation for real-time quantitative (RT-qPCR) analysis. The ChIP experiments were performed three times. Chromatin precipitated without an antibody constituted the negative control, while the isolated chromatin prior to precipitation was used as an input control. Primers used for ChIP-PCR are listed in S1 Table .
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