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18 protocols using fluorescently conjugated secondary antibodies

1

Western Blot Analysis of RNA-Binding Proteins

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Western blot analysis was performed using samples of whole cell lysate or eluate from immunoprecipitation prepared in NuPage sample buffer supplemented with antioxidant reagent according to manufacturer’s specifications (Invitrogen). Samples were heated (10’ @ 70°C), then separated by electrophoresis in NuPage 4–20% polyacrylamide gels and transferred to nitrocellulose membanes using the NuPage XCell II Blow Module (Invitrogen). Blots were visualized using the Li-COR Odyssey CLX fluorescent scanner after blocking with fluorescent blocking buffer (Li-COR Biosciences), incubation with primary antibodies and fluorescently conjugated secondary antibodies (Li-COR Biosciences).
Primary antibodies: Mouse monoclonal–HNRNPC1/C2 (sc-32308, Santa Cruz), HNRNPU (3C6, Millipore), SRSF3 (7B4, Millipore), SRSF1 (AK96, Millipore); Rabbit polyclonal–FUSIP1 (A302-282A-1,Bethyl), ILF2 (A303-147A-1, Bethyl), IgG (2729, Cell Signaling), U2AF1 (A302-079A-1, Bethyl), SAM68/KHDRBS1 (sc-333, Santa Cruz), SFRS10 (AV40528, Sigma).
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2

Western Blot Analysis of RFC4 and ACTB

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Total cellular proteins were extracted from tissues or cells, separated by SDS-PAGE, and transferred onto a polyvinylidene difluoride membrane (Pall, New York, USA). Membranes were blocked with 5% nonfat milk in 1% Tween-PBS (PBST) and then probed overnight with anti-RFC4 rabbit polyclonal antibody (1:1000, Epitomics, Burlingame, CA, USA) or anti-ACTB antibody (1:1000, Proteintech, Chicago, IL, USA). After three washing steps of 10 min in PBST, membranes were incubated with species-appropriate fluorescently-conjugated secondary antibodies (1:10000 in PBST, LI-COR Biosciences, Lincoln, NE, USA) for 1 h at room temperature. The immunoreactive signals were detected using the two-color fluorescent western blotting Odyssey infrared imaging system (LI-COR Biosciences).
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3

Western Blot Analysis of p53 in Cardiac Fibroblasts

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Protein was harvested from cardiac fibroblasts cultured in the presence or absence of serum (IMDM, 1X Penicillin/Streptomycin, +/− 10% FBS) for 48 hours at 37°C, 5% CO2. Concentration normalized protein was prepared with SDS loading buffer and run on a 12X Mini-Protean TGX gel (BioRad) at 300V for 25 min. The protein was transferred from the gel to a nitrocellulose membrane using the Trans-Blot Turbo System (BioRad). After blocking with TBST+3% cold fish gelatin, the membrane was probed using primary antibodies to p53 (Catalog#ab31333, Abcam) and alpha Tubulin (Catalog#T6199, Sigma), washed with TBST, and labeled using fluorescently conjugated secondary antibodies (LI-COR Biosciences). After washing with TBST, the membrane was visualized on an Odyssey scanner (LI-COR Biosciences). Densitometry analysis was performed using the Gel Analyzer unit of ImageJ software (NIH).
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4

Western Blot Protein Analysis Protocol

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Protein lysates were obtained after washing the cells with PBS and subsequently adding protein lysis buffer [50 mM Tris–Cl (pH 7.4), 100 mM NaCl, 1.0% IGEPAL® CA-630 (all from Sigma-Aldrich)] containing protease inhibitors Complete® and Pefabloc® SC (both from Merck). Lysates were snap frozen, thawed on ice and cleared by centrifugation at 13,000 RPM for 10 min at 4 °C. Lysates were mixed with 4 × sample buffer [8% SDS, 20% v/v glycerol, 0.002% bromophenolblue, 62.5 mM Tris–Cl (pH 6.8)] supplemented with 100 mM dithiothreitol (DTT) and incubated for 10 min at 95 °C. Proteins were separated on 4–15% Criterion TGX precast gels (Bio-Rad), and transferred to nitrocellulose membranes using the Trans-Blot® Turbo™ Transfer System (Bio-Rad). Blots were blocked using 5% non-fat dry milk (Blotto, Santa Cruz Biotechnologies) in PBS for 1 h at RT. Primary antibody incubations were performed overnight at 4 °C in blocking buffer. After washing in PBS, 0.1% v/v TWEEN® 20, blots were incubated for 1 h at RT with fluorescently conjugated secondary antibodies (LI-COR Biosciences or Jackson ImmuneResearch). After washing in PBS, 0.1% v/v TWEEN® 20, blots were imaged using the Odyssey CLx Imaging system and analysed with Image Studio™ Lite Ver 5.2 (both from LI-COR Biosciences).
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5

Wnt Protein Expression Analysis in Xenopus Embryos

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Embryos used for the analysis of Wnt protein expression were cultured and microinjected using the methods described above. Five YFP-positive embryos were flash-frozen at the mid gastrula stage (Nieuwkoop and Faber stage 10.5). Embryos were homogenized through a P200 pipet tip in 10 μl/embryo lysis buffer (20 mM Tris, pH 7.5, 1% Triton X-100, 140 mM NaCl, 10% glycerol, 10 mM EDTA, 1 mM DTT) supplemented with protease inhibitor cocktail (Sigma P8340, 1:100 dilution). Lysates were centrifuged at 1000 x g for 5 min to remove yolk protein, and subsequently at 14,000 rpm for 10 minutes to remove cellular debris. The lipid-free layer of supernatant was recovered and 1–2 embryo equivalents were loaded on to a 10% SDS-PAGE gel. Protein gels were run at 100 V and transferred to a nitrocellulose membrane for 2 h at 350 mA. Membranes were blocked for 1 h in bløk™-FL Fluorescent Blocker (Millipore), and probed with antibodies against β-tubulin (BD Pharmingen 556321, 1:1000), xWnt8 (see above, 0.14 μg/ml), Wnt1 (Abcam 15251, 1:1000), Wnt3a (Abcam 28472, 20 μg/ml) and Wnt5a (Cell Signaling Technology C27E8, 1:1000). Blots were incubated with fluorescently-conjugated secondary antibodies (LI-COR, 1:50,000) for 1 h, washed for at least 2 h (optimally overnight) in TBS-T, and visualized on a LI-COR Odyssey scanner.
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6

Protein Extraction and Western Blotting

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Equal amounts (around 0.2 g) of tissues and counted cells were homogenized in RIPA buffer (0.1 g/mL) (Beyotime, China) by using a Speed-Mill PLUS homogenizer (Analytik Jena, Jena, Germany). The homogenate was then centrifuged at 12,000 × rpm for 20 mins at 4 °C, and the supernatant proteins were collected. After the measurement of protein concentration, the proteins were boiled in 1× sodium dodecyl sulfate (SDS) sample buffer (100 μl for 1× 106 cells) for 5 mins, and then equal amounts of protein (50 μg) were loaded and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to polyvinylidene fluoride (PVDF) membrane (GE Healthcare, Buckinghamshire, UK). The membrane was then incubated with specific primary antibodies (Additional file 4: Table S2) and fluorescently conjugated secondary antibodies (Licor, USA), followed by the detection with the Odyssey system (TAITEC Co., Saitama, Japan).
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7

Western Blot Analysis of p53 in Cardiac Fibroblasts

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Protein was harvested from cardiac fibroblasts cultured in the presence or absence of serum (IMDM, 1X Penicillin/Streptomycin, +/− 10% FBS) for 48 hours at 37°C, 5% CO2. Concentration normalized protein was prepared with SDS loading buffer and run on a 12X Mini-Protean TGX gel (BioRad) at 300V for 25 min. The protein was transferred from the gel to a nitrocellulose membrane using the Trans-Blot Turbo System (BioRad). After blocking with TBST+3% cold fish gelatin, the membrane was probed using primary antibodies to p53 (Catalog#ab31333, Abcam) and alpha Tubulin (Catalog#T6199, Sigma), washed with TBST, and labeled using fluorescently conjugated secondary antibodies (LI-COR Biosciences). After washing with TBST, the membrane was visualized on an Odyssey scanner (LI-COR Biosciences). Densitometry analysis was performed using the Gel Analyzer unit of ImageJ software (NIH).
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8

Liver Protein Extraction and Western Blot

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Liver tissue samples were homogenized in RIPA buffer (50 mmol/L Tris-HCL, pH 8.0, 150 mmol/L NaCl, 1% NP-40, 0.5% w/v sodium deoxycholate, and 0.1% w/v sodium dodecyl sulfate) with 1× protease inhibitors and 1× protease/phosphatase inhibitors (Cell Signaling). Protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis using a 4%–12% bis-tris gradient gel (Bio-Rad) and transferred onto a nitrocellulose membrane. Membranes were incubated overnight against antibodies against GPAM (Novus Biologicals) and β-actin (Sigma), followed by fluorescently conjugated secondary antibodies (Licor) and detection on the Licor Odyssey. Immunoblot intensities were quantified using the Licor Image studio analysis software.
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9

Western Blot Analysis of Metabolic Enzymes

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Whole cell extracts were fractionated by SDS-PAGE and transferred to
nitrocellulose membrane by wet transfer for 4 h on ice at 60 V. Blots were
blocked in TBS blocking buffer (Li-Cor) and then stained overnight with
primary antibody at 4°C. The next day, membranes were washed three
times in TBST (TBS and 0.1% Tween-20), stained with fluorescently conjugated
secondary antibodies (Li-Cor) at 1:10,000 dilution in TBS blocking buffer
for 1 h at room temperature, and then imaged in the 680- and 800-nm
channels. Antibodies used: Gapdh (CST), Gpi1 (Thermo Fisher), Ldha (CST),
Pdha1 (Abcam), Tpi1 (Abcam), G6pdx (Abcam), a-tubulin (Santa Cruz).
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10

Quantitative Western Blot Analysis in Metabolic Tissues

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Mouse liver, white adipose and gastrocnemius skeletal muscle was lysed in homogenization buffer. Protein concentrations were determined using the Bradford assay (Thermo Fisher) before separation by SDS-PAGE. Proteins were transferred to nitrocellulose membranes (Licor) and blocked for 10 min in blocking buffer (Licor) diluted in TBST. Membranes were then incubated with Revert stain (Licor) per manufacturer’s instructions to quantitate total protein levels. Membranes were then incubated overnight with primary antibodies followed by a 60 min incubation at room temperature with fluorescently conjugated secondary antibodies (Licor). The following primary antibodies from Cell Signaling Technologies were used at a 1:1000 dilution: phosphorylated AKT (S473), phosphorylated GSK3β (S9) and GSK3β. The following primary antibodies from ProteinTech were used at a 1:1000 dilution: AKT, SOD1, SOD2 and catalase. Fluorescent intensity was quantified using Image Studio (Licor).
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