Zebrafish embryos at 5 dpf or freshly dissected adult ventricular tissues were collected in 1.5-ml safe-lock tubes (Eppendorf) and mechanically homogenized (Blender tissue homogenizer; Next Advance Inc.) in radioimmunoprecipitation assay lysis buffer (Sigma-Aldrich) containing proteinase inhibitor, 1 mM phenylmethylsulfonyl fluoride, and stainless steel beads. Standard protocols of Western blotting were followed. For each sample, >20 μg of total protein was loaded on an SDS polyacrylamide gel. The following primary antibodies were used: anti-EGFP (1:2000) (Santa Cruz Biotechnology), anti-mCherry (1:1000; Novus Biologicals), anti–ZO-1 (1:1000; Invitrogen), and anti-cleaved caspase-3 (1:1000; Cell Signaling Technology). Anti-actin (1:2000) (Sigma-Aldrich) and anti-GAPDH (1:2000; Santa Cruz Biotechnology) were used as an internal control.
Anti egfp
Manufactured by Santa Cruz Biotechnology
Sourced in China, United States
Anti-EGFP is an antibody produced by Santa Cruz Biotechnology that specifically binds to the Enhanced Green Fluorescent Protein (EGFP). This antibody can be used to detect and localize EGFP in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared imaging system, by LI COR (3 mentions)
Anti actin, by Merck Group (2 mentions)
Infrared dye coupled secondary antibodies, by LI COR (2 mentions)
Sc 9996, by Santa Cruz Biotechnology (2 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Safe lock tube, by Eppendorf (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Merck Group (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti mcherry, by Novus Biologicals (1 mentions)
Anti zo 1, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by ABclonal (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Nonidet p, by Roche (1 mentions)
Las 3000 imaging system, by Fujifilm (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Ecl plus western blotting detection kit, by GE Healthcare (1 mentions)
Anti β actin, by Abcam (1 mentions)
Hrp conjugated anti mouse igg, by Cell Signaling Technology (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Anti ago1, by Merck Group (1 mentions)
12 protocols using anti egfp
1
Zebrafish and Ventricular Tissue Protein Analysis
2
Nras61K Oncogene Expression in Zebrafish
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The 293 T cells were collected after trypsinization and resuspended in mammosphere medium. The cells were then transfected with 100 nmol/liter pAc-GFP-nras61K or pAc-GFP as control. The liver tissues from the 3, 6, 9 and 12 mpf Tg(fabp10:nras61K) transgenic zebrafish and WT (12 mpf) were collected respectively, and the livers of four fish were pooled to generate one sample. The samples were lysed with RIPA lysis buffer [20 mM Tris/HCl (pH 7.4), 150 mM NaCl, 0.5% Nonidet P40 and 1 × protease inhibitor cocktail (Roche, Penzber, Germany)], and the lysates were incubated with the suitable antibody including anti-β actin (AB clonal, Wuhan, China, AE012), anti-EGFP (Santa Cruz, CA, SC-47778), anti-MEK1, anti-MEK2, anti-ERK, anti-pMEK1/2, anti-pERK.
3
Western Blot Analysis of CYP3A4/7 and EGFP
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HepaRG WT and C3 cells at D7d, and C87 HepG2 cells were suspended in RIPA buffer (Sigma-Aldrich) and placed on a rotator for 30 min at 4°C. Proteins were collected by centrifugation at 12,000 rpm for 20 min at 4°C. Thereafter, 10–20 µg of protein mixed with 25% LDS sample buffer (NuPAGE) was separated by electrophoresis on a 5–20% SDS-polyacrylamide gradient gels (ATTO) and transferred to a polyvinylidene fluoride membrane. For each of sample, three western blots were prepared. Next, the membranes were pre-hybridized with PBS containing 3% skimmed milk (Difco, BD) overnight at 4°C, and then incubated with the primary antibody diluted in PBS containing 3% skimmed milk for 1 h at room temperature. First, the primary anti-CYP3A4/7antibody (rabbit polyclonal, 1∶500; Enzo) was used. The hybridized bands were visualized using the ECL Plus Western Blotting Detection Kit (GE Healthcare). Second, the membrane was co-incubated with two primary antibodies, namely, anti-EGFP (mouse monoclonal, 1∶200; Santa Cruz Biotechnology, Inc.) and anti-β-actin (mouse monoclonal, 1∶5,000; Abcam). The relative expression levels of CYP3A4/7 and EGFP in cells were calculated by normalizing the intensities of these bands to that of β-actin using an LAS-3000 imaging system (Fujifilm, Tokyo, Japan).
4
Western Blot Analysis of Cellular Proteins
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Cultured U2OS cells transfected with corresponding plasmid DNA (or U2OS cytoplasmic or nuclear lysates) were lysed in Laemmli loading buffer (250 mM Tris–HCl pH 6.4; 2% SDS; 100 nM b-MeEtOH), separated SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked in Odyssey blocking buffer (LI-COR) or 5% non-fat milk containing 1xPBST for 1 h at RT. Incubation with anti-FLAG (Rockland, cat.200-301-383 S), anti-LaminB1 + B2 (Abcam, clone X223), anti-AGO1 (Sigma-Aldrich, clone 4B8), anti-EGFP (Santa Cruz, clone B-2), anti-α-tubulin (Sigma-Aldrich, clone AA13), anti-β-actin (Santa Cruz, clone C-4), anti-GAPDH (BioLegend, clone FF26A/F9) was performed at 4 °C overnight in a 1:1000 dilution. After incubation with primary antibodies, membranes were washed twice in 1 × PBST for 10 min and incubated for 1 h at RT with infrared fluorescent anti-mouse IgG (Pierce, cat.SA5-10176) or anti-rat IgG (Pierce, cat.SA5-10024) diluted 1:10000 in Odyssey blocking buffer (LI-COR); or HRP-conjugated anti-mouse IgG (Cell Signaling, cat. 7076) diluted 1:1000 in 5% non-fat milk containing 1xPBST. Membranes were washed four times for 10 min in PBST and signals were then acquired with Odyssey infrared imaging system (LI-COR) or luminescent detection.
5
Western Blot Analysis of HIV-1 p24 and eGFP
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Virion and cell lysates were separated on 4–15% polyacrylamide gels and transferred to Immobilon-FL membranes. Anti-p24 (183-H12-5C, NIH AIDS Reagent Program), anti–eGFP (Santa Cruz) and infrared dye coupled secondary antibodies (LI-COR) were used for immunoprobing. Scanning was performed with the Odyssey infrared imaging system (LI-COR) in accordance with the manufacturer’s instructions at 700 or 800 nm, accordingly.
6
Comprehensive Protein Expression Analysis
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Western blot was performed similarly to previously described methods [34 (link)], using anti-CtBP2 (1:500), anti-p16INK4A (1:1000), anti-PCNA (1:1000), anti-EGFP (1:1000), anti-CDK2 (1:500), anti-CDK4 (1:500), anti-CyclinD (1:500), anti-CyclinE (1:500), anti-p21 (1:1000), anti-Bax (1:1000) anti-E-cadherin (1:1000), anti-vimentin (1:1000), anti-MMP-2 (1:500) and anti-Glyceraldehyde-3-phosphate dehydro-genase (GAPDH) (1:1000, all the above antibodies from Santa Cruz Biotechnology, America). ImageJ (NIH) was used to compare the density of bands on western blot. Mean densitometry data from independent experiments were normalized by GAPDH.
7
Western Blot Analysis of Protein Expression
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Samples were placed in six-well plates and collected in 250 μl of lysis buffer that included NP-40 and protease inhibitors as mentioned earlier, and sample buffer is added at 20% of the total volume. After SDS-PAGE, protein bands were transferred to nitrocellulose membrane (Bio-Rad). Primary antibodies include anti-PLCβ1 (Santa Cruz, sc-5291), anti-Ago2 (Abcam, ab32381), anti-actin (Abcam, ab8226), and anti-eGFP (Santa Cruz, sc-8334). Membranes were treated with antibodies diluted 1:1000 in 0.5% milk, washed 3 times for 5 min, before applying secondary antibiotic (anti-mouse or anti-rabbit from Santa Cruz) at a concentration of 1:2000. Membranes were washed 3 times for 10 min before imaging on a Bio-Rad chemi-doc imager to determine the band intensities. Bands were measured at several sensitivities and exposure times to ensure the intensities were in a linear range. Data were analyzed using ImageJ.
8
Immunoblot Analysis of Viral Proteins
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Virion and cell lysates were separated on 4–15% polyacrylamide gels and transferred to Immobilon-FL membranes. The blots were probed with anti-p24 (183-H12-5C, NIH AIDS Reagent Program), anti-eGFP (Santa Cruz), and infrared dye coupled secondary antibodies (LI-COR) were used for immunoprobing. Scanning was performed with the Odyssey infrared imaging system (LI-COR) in accordance with the manufacturer’s instructions at 700 or 800 nm, accordingly.
9
Quantitative Western Blot Analysis
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HuH7, C2, and NSC34 cell lysates were prepared using 10 mM PBS (pH 7.4) containing 1% of Triton X-100 and protease inhibitors (Roche Diagnostics). Western blot on mouse plasma was performed on samples diluted 1:4 in distilled water. Homogenates of mouse tissues were prepared as indicated for GAA activity. Protein concentration was determined using the BCA protein assay (Thermo Fisher Scientific). SDS-PAGE electrophoresis was performed in a 4%–12% polyacrylamide gel. After transfer, the membrane was blocked with Odyssey buffer (LI-COR Biosciences) and incubated with an anti-GAA antibody (rabbit monoclonal, clone EPR4716(2), Abcam), anti-EGFP (mouse monoclonal, sc-9996, Santa Cruz Biotechnology), anti-tubulin (mouse monoclonal, clone DM1A, Sigma-Aldrich), or anti-Gapdh (rabbit polyclonal, PA1-988, Thermo Fisher Scientific). The membrane was washed and incubated with the appropriate secondary antibody (LI-COR Biosciences) and visualized with the Odyssey imaging system (LI-COR Biosciences). For western blot quantification, we used either ImageJ or Image Studio Lite 4.0. The quantification of the hGAA protein bands in mouse tissues was normalized using either tubulin or Gapdh bands. The quantification of the hGAA protein band in plasma was normalized using a non-specific band detected by the anti-hGAA antibody in mouse plasma (used as a loading control).
10
Protein Extraction and Western Blot Analysis
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Mycelium was grown for 3–5 days in liquid medium, dried and grounded in liquid nitrogen. Pulverized mycelium mixed with extraction buffer (100 mM Tris-HCl pH 7.6, 250 mM NaCl, 10% glycerol, 0.2% NP-40, 2 mM EDTA, 2 mM DTT) containing protease inhibitor cocktail IV (1:100, Roche, 04693132001) and 1 mM PMSF. Protein samples were separated by SDS-PAGE [58 (link)] and transferred onto nitrocellulose membranes by Western blot [59 (link)]. For immunodetection anti-FLAG (Sigma-Aldrich, F3165, 1:12000), anti-HA (Sigma-Aldrich, H9658, 1:3000) and secondary antibody anti-Mouse (Dianova, 115-035-003, 1:10000) were used. Anti-eGFP (Santa Cruz Biotechnology, sc-9996, 1:4000) antibody was used for expression control of eGFP-tagged kinases. Signals were detected using Immobilon Western Kit (Millipore, WBKLS0500).
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