Total protein extractions were performed as previously described [59 (link)] Nuclear/cytoplasmic protein fractionations were performed using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo, Waltham, MA, USA), according to manufacturer's instructions. Histone extraction [60 (link)] and immunopreciptiation [59 (link)] experiments were performed as previously described. Western blot analyses were performed according to standard protocols. Antibodies used were anti-ATM S1981p (Rockland, Philadelphia, PA, USA), anti-p53 DO-1, anti-p-p53 (Ser15), anti-ATM 2C1 (Santa Cruz), anti-RanBP9 (Abcam, Cambridge, MA, USA), anti-pChk2 (Thr68), anti-Chk2, anti-γH2AX, anti-H2A, anti-PARP, anti-GAPDH-HRP-conjugated, anti-β-actin (Cell Signaling Technology, Danvers, MA, USA) and anti-p84 (GeneTex, San Antonio, TX, USA). ImageQuant software (Biorad, Hercules, CA, USA) was used for the quantification of western blot data.
Imagequant software
Manufactured by Bio-Rad
Sourced in United States
ImageQuant software is a digital image analysis tool for quantifying signals from gel-based and membrane-based assays, including Western blots, Northern blots, and other electrophoretic techniques. The software provides tools for image acquisition, processing, and data analysis.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh antibody, by Huabio (2 mentions)
Caspase 3 antibody, by OriGene (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Sds page sample loading buffer, by Beyotime (2 mentions)
Protease inhibitors, by Dingguo (2 mentions)
Anti p53 do 1, by Santa Cruz Biotechnology (1 mentions)
Anti chk2, by Cell Signaling Technology (1 mentions)
Anti pchk2 thr68, by Cell Signaling Technology (1 mentions)
Anti atm s1981p, by Rockland Immunochemicals (1 mentions)
Anti atm 2c1, by Santa Cruz Biotechnology (1 mentions)
Anti ranbp9, by Abcam (1 mentions)
Anti p84, by GeneTex (1 mentions)
Anti h2a, by Cell Signaling Technology (1 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Anti γh2ax, by Cell Signaling Technology (1 mentions)
Anti parp, by Cell Signaling Technology (1 mentions)
Anti grb2, by Cell Signaling Technology (1 mentions)
Anti actin antibody, by Merck Group (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
15 protocols using imagequant software
1
Protein Extraction and Western Blot Analysis
2
Western Blot Quantification of DNA Repair Proteins
3
Western Blot Analysis of dfsAC and NKA
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Tissue samples were processed for western blots in a manner similar to Roa et al. (2014 ) and Roa and Tresguerres (2016 ). In brief, 20 μg of proteins were separated on 7.5% polyacrylamide mini gel (60 V 15 min, 200 V 45 min) and transferred to a polyvinylidene difluoride (PVDF) membrane (Bio‐Rad). After transfer, PVDF membranes were incubated in blocking buffer (tris‐buffered saline, 1% tween, 5% skim milk) at room temperature (RT) for 1 h and incubated in the primary antibody at 4°C overnight (anti‐dfsAC = 3 μg/mL; anti‐NKA = 1.5 μg/mL). PVDF membranes were washed 3× (10 min each) in tris‐buffered saline + 1% tween (TBS‐T), incubated in the appropriate anti‐rabbit or anti‐mouse secondary antibodies (1:10,000) at RT for 1 h, and washed 3× (10 min each) in TBS‐T. Bands were made visible through addition of ECL Prime Western Blotting Detection Reagent (GE Healthcare, Waukesha, WI) and imaged and analyzed in a BioRad Universal III Hood using ImageQuant software (BioRad). PVDF membranes incubated in blocking buffer with anti‐dfsAC antibodies and 300‐fold excess blocking peptide served as control and did not show any bands.
4
Quantitative Western Blot Analysis
5
Immunoblotting Analysis of Filamin B
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MEFs were cultured with or without PMA in serum-starved condition overnight. Flnb+/+ and Flnb−/− tumor tissues were homogenized and immunoblotted as described earlier,10 (link) followed by incubation with primary antibody against FLNB (Chemicon International, Inc., Temecula, CA, USA), FLNA (EMD Millipore), Actin (Sigma Aldrich), phosphorylated ERK1/2, total ERK1/2, phosphorylated-PKC-α/β and phosphorylated-PKC-δ/θ (Cell Signaling Technologies, Danvers, MA, USA) and MMP-9 (Nordic BioSite, Taby, Sweden). Membranes were then incubated with an appropriate anti-mouse or anti-rabbit IgG-horseradish peroxide conjugated secondary antibody (Amersham Biosciences, Amersham, UK) and the proteins were visualized by using enhanced chemiluminescence (Amersham Biosciences). The densities of bands were quantified by ImageQuant software (Bio-Rad, Hercules, CA, USA). Actin was used as an internal loading control.
6
Quantitative Immunoblotting Analysis of Cellular Signaling
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Protein lysates from cells were assayed by immunoblotting as described earlier.11 (link) Primary antibodies directed against FLNA (Bethyl Laboratories), p-AKT, AKT, p-ERK1/2, ERK1/2, MYCN, p-MEK, p-STAT3, p-NF-κB, p53, cleaved caspase 3 (Cell Signaling), and Actin (Sigma-Aldrich) were used. The densities of triplicate bands were quantified by ImageQuant software (Bio-Rad). Expression levels of FLNA, p53, MYCN and cleaved caspase 3 were normalized to Actin, whereas expression levels of p-AKT and p-ERK1/2, p-STAT3, p-NFκB were normalized to their respective total proteins.
7
Western Blotting Analysis of Neurodegenerative Markers
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For the western blotting analysis, after dissecting the SN and striatum from all brain tissues, these regions were lysed on ice in RIPA buffer (50 mM Tris/HCl, pH 8.0, 150 mM NaCl, 2 mM sodium orthovanadate, 1% Nonidet-P40, 0.5% sodium deoxycholate, 0.1% SDS, and 1% of a protease inhibitor mixture containing AEBSF, pepstatinA, E-64, bestatin, leupeptin, and aprotinin). The soluble fraction was obtained and equal amounts of total cell lysates were loaded in each lane of a 10% polyacrylamide gel. After electrophoresis and transfer onto a polyvinylidene difluoride (PVDF) membrane, specific protein bands were detected using appropriate primary antibodies (rabbit anti-Pitx3; rabbit anti-Nurr1; mouse anti-TH and mouse anti-GAPDH and respective secondary antibodies conjugated to horseradish peroxidase (anti-rabbit or anti-mouse) followed by Enhanced Chemiluminescence (ECL) detection. Densitometric analyses were performed by using the ImageQuant software (Bio-Rad).
8
Immunoblot Analysis of Cellular Proteins
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Cell lysates were separated by gel electrophoresis on 12.5% or 15% SDS-polyacrylamide gels and transferred to Immobilon polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA) for 90 min at 100 V in a Miniprotean III transfer tank (Bio-Rad, Hercules, CA). Immunoblots were incubated with anti-core antibody at a dilution of 1/10,000, followed by incubation with alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin (Jackson ImmunoResearch, West Grove, PA) at a dilution of 1/20,000. Immunoblots were imaged on a PhosporImager (Bio-Rad), and band quantitation was conducted with ImageQuant software (Bio-Rad).
9
Western Blot Analysis of Protein Lysates
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Cells and tissues were lysed in ice-cold lysis buffer containing 25 mM Tris (pH7.5), 2 mM MgCl2, 600 mM NaCl, 2 mM EDTA, 0.5% Nonidet P-40, and 1 × protease and phosphatase mixture inhibitors (Roche). 20 μg aliquots of lysate were separated by electrophoresis on a 10% SDS-polyacrylamide gel and transferred on a polyvinylidene difluoride (PVDF) membrane (Amersham). Incubation with primary antibody was performed overnight in 5% milk-TBS-Tween. The next day, membranes were incubated at room temperature for 1 h with HRP-linked secondary antibodies. Western-blot revelation was performed using ECL (Amersham). Imaging and analysis were performed on a chemidoc imager (Bio-rad) using the ImageQuant software. Images presented in the figures have been cropped around the expected bands for figure design purpose. The original (raw) data for Fig. 1a and Supplementary Figure 1J and including molecular weight markers is presented in Supplementary Figure 10 .
10
Quantitative Western Blot Analysis of Neprilysin
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