2 × 105 cells were lysed in a lysis buffer (aqua dest. + 10 mM NaCl/10 mM Tris (hydroxymethyl)-aminomethan/3 mM MgCl2/5 % NP-40) and proteins were resolved by SDS-PAGE and identified by immunoblotting. We used an affinity-purified polyclonal rabbit anti-peptide antibody directed against the C terminus of human SEC62 (self-made), a polyclonal rabbit antibody directed against the C terminus of human SOX2 (abcam pic, Cambridge, UK) and a monoclonal mouse antibody directed against the N terminus of human b-actin (Sigma-Aldrich Co., St. Louis, MO, USA). The secondary antibodies used were ECL Plex goat anti-rabbit Cy5 or anti-mouse Cy3 conjugates (GE Healthcare, Munich, Germany). Blots were imaged using the Typhoon-Trio system and Image Quant TL software 7.0 (GE Healthcare, Munich, Germany). The SEC62, SOX2 and β-actin levels were quantified and normalized against β-actin.
Image quant tl software 7
Manufactured by GE Healthcare
Sourced in Germany
The Image Quant TL software 7.0 is a quantification and analysis tool for gel and blot imaging. It provides features for capturing, processing, and analyzing digital images of gels, blots, and other electrophoresis-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Typhoon trio system, by GE Healthcare (2 mentions)
Ecl plex goat anti rabbit cy5, by GE Healthcare (2 mentions)
Anti mouse cy3 conjugates, by GE Healthcare (2 mentions)
Typhoon trio imaging system, by GE Healthcare (2 mentions)
Monoclonal mouse antibody, by Merck Group (1 mentions)
Anti human β actin, by Merck Group (1 mentions)
Anti human gapdh sc 25778, by Santa Cruz Biotechnology (1 mentions)
Benchmark pre stained protein ladder, by Thermo Fisher Scientific (1 mentions)
6 protocols using image quant tl software 7
1
Quantitative Immunoblotting of SEC62, SOX2 and β-Actin
2
Western Blot Imaging and Analysis
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Western blots were scanned and analysed using the Typhoon-Trio imaging system (GE Healthcare) and the Image Quant TL software 7.0 (GE Healthcare). The primary antibodies were visualized using ECL Plex goat anti-rabbit IgG-Cy5 conjugate or ECL Plex goat antimouse IgG-Cy3 conjugate (GE Healthcare, Freiburg, Germany). Supplementary Table 1 lists primary antibodies used in this study. Full Western blot scans are shown in Supplementary Fig. 4 .
3
Immunoblotting of Cellular Proteins
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2 × 105 HeLa cells were lysed in a lysis buffer (aqua dest. + 10 mM NaCl/10 mM Tris(hydroxymethyl)-aminomethan/3 mM MgCl2/5 % NP-40) and proteins were resolved by SDS-PAGE and identified by immunoblotting. Antibodies used were the previously described anti-human Sec62, monoclonal anti-human β-actin (Sigma-Aldrich Co., St. Louis, MO, USA), anti-human E-cadherin Clone 24E10 (Cell signaling Technology, Cambridge, UK), anti-human vimentin Clone V9 (Dako Denmark A/S, Glostrup, Denmark) and anti-human GAPDH (sc-25778, Santa Cruz Biotechnology, Dallas, TX, USA) antibody. Secondary antibodies used were ECL Plex goat anti-rabbit Cy5 or anti-mouse Cy3 conjugates (GE Healthcare, Munich, Germany). Blots were imaged with the Typhoon-Trio system and the Image Quant TL software 7.0 (GE Healthcare, Munich, Germany). Sec62, vimentin, and β-actin levels were quantified and normalized to GAPDH.
4
Membrane Protein Extraction and Phosphotyrosine Analysis
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Membrane/cytoskeleton proteins were extracted from pelleted membranes with 4 to 6 volumes of deoxycholate (DC) buffer: 1% DC in 50 mM Tris-HCl, 150 mM NaCl, and pH 8.1.34 (link) Ten μg of proteins from each sample and 10 μL of BenchMark Pre-Stained Protein Ladder (Invitrogen, ThermoFisher Scientific) were loaded on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, Mini-PROTEAN TGX gels, 4%-15%; BIO-RAD). WB were performed on polyvinylidene fluoride (PVDF) membranes against pY-containing proteins (pY PY99 1/6000 from Santa Cruz Biotechnology, Dallas, TX). Washing and incubation with primary and secondary antibodies were done in tris-buffered saline (TBS-T: 1× TBS + 0.05% Tween-20) and Top Block buffer (Sigma-Aldrich, 4% in TBS-T), respectively. Secondary antibody (polyclonal goat antimouse immunoglobulins HRP, Dako) was diluted at 1/6000 or 1/10 000 in Top Block buffer. Bands of interest (from WB and Ponceau red staining) were quantified by densitometry (ImageQuant TL software 7.0; GE Healthcare, Uppsala, Sweden) and expressed as “volume.” pY signals were normalized by total protein loading.
5
Phosphoprotein Detection by Pro-Q Diamond
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Detection of phosphoproteins was accomplished with the Pro-Q ® Diamond Phosphoprotein Blot Stain Kit (P33356) from Molecular Probes (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. Briefly, proteins were fixed on the PVDF membrane, washed, stained with Pro-Q ® Diamond Blot Stain Reagent and destained. After drying the blot, fluorescence was detected with the Typhoon-Trio imaging system (GE-Healthcare) using the 580-nm band-pass filter (580 BP 30) in combination with the Image Quant TL software 7.0 (GE-Healthcare).
After staining of phosphoproteins, total proteins were stained with Ponceau S solution (0.2% (w/v) Ponceau S, 15% (v/v) acetic acid, 40% (v/v) methanol). After washing, the blot was blocked and incubated with antibodies as described in 2.9.
After staining of phosphoproteins, total proteins were stained with Ponceau S solution (0.2% (w/v) Ponceau S, 15% (v/v) acetic acid, 40% (v/v) methanol). After washing, the blot was blocked and incubated with antibodies as described in 2.9.
6
Quantifying 11C-Sugars in Plant Tissues
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Soluble 11 C-sugars were separated and analyzed by TLC (Babst et al., 2013) . Glass backed NH 2 -silica high-performance TLC plates (200 lm, w/UV254) were used for the sugar separation (Sorbent Technologies, http://www.sorbtech.com/). Plates were pre-spotted with sugar standards of glucose, sucrose and fructose for registration of 11 C-sugar R f signatures against those of authentic compounds and then with 1-and 2-ll aliquots of radioactive leaf extract using a semi-automatic Linomat 5 sample applicator (Camag Scientific, http://www.camag.com/) for high precision of spot size and sample volume. The larger volume of extract was sometimes needed to visualize the 11 C-hexose sugars, which were usually at a lower concentration than sucrose. The TLC plates were developed using a mobile phase consisting of 75:25 acetonitrile: water (v/v). Developed plates were imaged using autoradiography (Typhoon FLA 7000) to determine the fraction of each radiolabeled sugar. IMAGEQUANT TL software 7.0 (GE Healthcare Bio-Sciences) was used to analyze both the radiographic images to determine the relative amount of 11 C within the individual sugars. These values were summed across the soluble sugar pool, and presented as the total 11 C-sugars corrected to reflect the fraction of the total 11 C-activity within the targeted tissue using 11 C-soluble and 11 Cinsoluble fractions.
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