Western blot analysis was performed as described previously.29 Briefly, aliquots of total protein (10 µg) were electrophoresed on sodium dodecyl sulfate polyacrylamide, 10% Tris‐HCl gels (Bio‐Rad Laboratories). The separated proteins were transferred to polyvinylidene difluoride membranes (Bio‐Rad Laboratories) and incubated with primary antibodies overnight at 4°C. Proteins were detected using the following antibodies: anti‐VE‐cadherin (Abcam), anti‐ZO‐1, anti‐E‐cadherin, anti‐Zinc Finger E‐Box Binding Homeobox 1 (ZEB1), anti‐N‐cadherin, anti‐Snail (Cell Signaling Technology, Danvers, MA, USA), anti‐CD13, anti‐CD133, anti‐EpCAM (Abcam) and anti‐β actin (Sigma, Tokyo, Japan).
Tris hcl gel
Manufactured by Bio-Rad
Sourced in United States
4–15% Tris-HCl gels are a type of polyacrylamide gel used for electrophoresis. They feature a gradient of 4% to 15% polyacrylamide concentration, which allows for the separation of a wide range of protein molecular weights.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Bio-Rad (7 mentions)
Bca assay, by Thermo Fisher Scientific (5 mentions)
Trans blot turbo transfer system, by Bio-Rad (5 mentions)
Anti β actin, by Merck Group (4 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (4 mentions)
Cell lysis buffer, by Cell Signaling Technology (4 mentions)
Ripa buffer, by Boston BioProducts (4 mentions)
Nitrocellulose membrane, by Bio-Rad (3 mentions)
P akt, by Cell Signaling Technology (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Ripa buffer, by Thermo Fisher Scientific (3 mentions)
Chemidoc imaging system, by Bio-Rad (3 mentions)
Clarity western ecl substrate, by Bio-Rad (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Ripa buffer, by Merck Group (3 mentions)
Chemidoc xrs system, by Bio-Rad (3 mentions)
Radioimmunoprecipitation assay buffer, by Thermo Fisher Scientific (2 mentions)
Las 4000, by Fujifilm (2 mentions)
Adiponectin, by BD (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
93 protocols using tris hcl gel
1
Western Blot Protein Analysis Protocol
2
Western Blotting of RecA Protein
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Cells were pelleted and then resuspended in 1xSDS sample buffer and heated to 95°C for 5 min. Equal amounts of total protein were run on 10% Tris-HCl gels (Bio-Rad) at 150V for separation. Resolved proteins were transferred to polyvinylidene fluoride membranes and probed with 1:5000 dilution of primary antibodies against RecA (Sigma) and secondary horseradish-peroxidase-conjugated antibody (1:5000). Blots were visualized using a FluorChem M imager (ProteinSimple).
3
Western Blot Analysis of PopZ and RpoA
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Cells were pelleted and then resuspended in SDS sample buffer and heated to 95°C for 5 min. Equal amounts of total protein were run on 10% Tris-HCl gels (Bio-Rad Laboratories, Inc.) at 150 V for separation. Resolved proteins were transferred to polyvinylidene fluoride membranes and probed with 1:5,000 dilution of primary antibodies against PopZ (G. Bowman, University of Wyoming, Laramie, WY; Bowman et al., 2008 (link)) or RpoA (Sigma-Aldrich) and secondary horseradish peroxidase–conjugated antibody (1:5,000). Blots were visualized using a FluorChem M imager (ProteinSimple).
4
Quantitative Western Blot Analysis
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Western blotting was performed as described previously.24 Briefly, total protein was extracted from PDAC cell lines in radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Rockford, IL, USA) and nuclear proteins were extracted with the Nuclear Extraction Kit according to the manufacturers’ protocol. Aliquots of total protein (12 μg) were electrophoresed on sodium dodecyl sulfate polyacrylamide gels, 10% Tris‐HCL gels (Bio‐Rad Laboratories, Hercules, CA, USA). The separated proteins were transferred to polyvinylidene difluoride membranes (Bio‐Rad Laboratories) and incubated with primary antibodies for 1 hour. Proteins were detected with anti‐HDAC1 antibody (1:200 dilution; Santa Cruz Biotechnology), anti‐SNAIL antibody (1:2000 dilution; Abcam), anti‐ZEB1 antibody (1:1000 dilution; Santa Cruz Biotechnology), anti‐Cytokeratin 19 antibody (1:200 dilution; Santa Cruz Biotechnology), anti‐Histone H3 (1:2000; Cell Signaling Technology, Danvers, MA, USA) and anti–β‐actin antibody (diluted 1:4000; Sigma, Tokyo, Japan). The expression relative to actin expression was depicted as a column and measured with ImageJ software (rsb.info.nih.gov/ij).
5
Immunoblotting Analysis of Neuronal Markers
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TBS extracts were normalized to protein levels (BCA assay, Thermo Fisher), diluted in reducing sample buffer, size-fractionated by SDS-PAGE on 10% Tris-HCl gels (Bio-Rad), and transferred onto 0.2 μm nitrocellulose membranes. Immunoblots were probed with β-III-tubulin (Sigma Aldrich) and GAPDH (Cell Signaling) antibodies and visualized using chemiluminescence reagents (Pierce) followed by exposure onto autoradiography film (Gene Mate). Band densities were measured using Fiji software (NIH). TBS extracts for Casp2 detection were run on 10.5–14% gradient gels (Bio-Rad) and visualized using a Casp2 specific antibody (Abcam).
6
SLC1A5 Protein Expression Analysis
7
FABP4 and Adiponectin Protein Analysis
8
FABP4 and Adiponectin Protein Analysis
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Cells were harvested in cell lysis buffer (Cell Signaling) with 5% SDS and protease inhibitors (Pierce). 5–10 μg total protein was resolved in 10% Tris-HCl gels (Biorad) and transferred to PVDF membranes. Membranes were probed for FABP4 (a gift from Dr. Judith Storch at Rutgers University) and adiponectin (BD Biosciences), Chemiluminescence images were captured using an Imager (LAS 4000, Fuji).
9
Protein Complex Characterization by Gel Filtration
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Recombinant murine SPARC, CCL21, CCL19, CXCL10 (5ug each, R&D Systems or Peprotech) or BSA (Bovine Serum Albumin) (Sigma, St. Louis, MO) were resuspended in 70μL phosphate buffered saline (PBS) with 2 mM Calcium, mixed in the pairs indicated in Fig. 4 , and filtered using Sephadex G50 at 4 °C. Protein standards composed of BSA, ovalbumin (Sigma, St. Louis, MO), trypsin (Corning, Glendale, AZ), and CCL19 were also run through the column individually. Absorbance of eluted fractions at 280 nm were recorded and fractions that contained protein were pooled and concentrated with Amicon filters (Millipore, Billerica, MA). Concentrated eluate was mixed with Laemmli buffer and subjected to SDS-PAGE on 10% Tris–HCL gels (Biorad, Hercules, CA) followed by western blot onto nitrocellulose membranes on a semi-dry blotter (Biorad, Hercules, CA). Membranes were blocked with 5% nonfat dry milk then probed with anti-SPARC (R&D Systems), anti-CCL21 (Peprotech), anti-CCL19 (R&D Systems) or anti-CXCL10 (R&D Systems) antibodies and corresponding secondary antibodies (anti-rabbit 680 or anti-goat 800, Li-COR Biosciences). Blots were imaged using an Odyssey Infrared Imaging system (Li-COR Biosciences).
10
Protein Characterization by Mass Spectrometry
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