Protein samples were separated on 12% SDS-PAGE gels. After electrophoresis, the proteins were transfer to a nitrocellulose membrane (pore size 0.2 µm) with a Bio-rad Trans-Blot Cellin Towbin transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol), at 60 V, overnight. Membranes were blocked with 2% nonfat milk in PBS-T buffer (144 mg/L KH2PO4, 9 g/L NaCl, 795 mg/L Na2HPO4, pH 7.4, 0.1% Tween 20) for 1 hour. Membranes were probed with primary and secondary antibodies diluted in 2% milk in PBS-T as follows: TAg (pAb416) at 1:5,000 dilution (Harlow et al., 1986 (link)); GAPDH (Abcam ab9484) at 1:10,000; caveolin 1 (Santa Cruz SC-894) at 1:20,000; caveolin 2 (Cell signaling #8522) at 1:5,000; Clathrin heavy chain (Thermo Fisher Scientific, MA1-065) was kindly provided by Christiane Wobus (University of Michigan), used at 1:10,000, horseradish peroxidase (HRP)-conjugated ECL sheep anti-mouse (GE healthcare NA931V) at 1:5,000; and HRP-conjugated ECL donkey anti-rabbit antibody (GE healthcare NA934V) at 1:5,000. Protein bands were further visualized with HRP substrate (Millipore, WBLUF0100) and exposure to films.
Wbluf0100
Manufactured by Merck Group
Sourced in China, Germany
The WBLUF0100 is a laboratory centrifuge designed for routine sample separation and preparation. It features a compact design, adjustable speed settings, and a rotor capable of handling multiple sample tubes simultaneously. The core function of this equipment is to provide a controlled centrifugal force to separate components within liquid samples.
Automatically generated - may contain errors
Lab products found in correlation
Ipvh00010, by Merck Group (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Las 4000 mini, by GE Healthcare (2 mentions)
β actin, by Merck Group (2 mentions)
Human phospho kinase antibody array, by R&D Systems (2 mentions)
Caveolin 2, by Cell Signaling Technology (1 mentions)
Hrp conjugated ecl donkey anti rabbit antibody, by GE Healthcare (1 mentions)
Caveolin 1, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Abcam (1 mentions)
Anti β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Ecl reagent, by Keygen Biotech (1 mentions)
Mcherry ab167453, by Abcam (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Forte western hrp substrate, by Merck Group (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Luminata classico, by Merck Group (1 mentions)
Mini protean tgx precast protein gel, by Bio-Rad (1 mentions)
10 protocols using wbluf0100
1
Western Blot Analysis of Cellular Proteins
2
Quantitative Western Blot Analysis of TIMP-1 in hPASMCs
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Western blot was performed as previously described (19 (link)). Briefly, hPASMCs were washed in PBS and lysed in RIPA buffer supplemented with protease inhibitors on ice for 30 min. The supernatant was collected by centrifugation for 30 min with a speed of 12,000 rpm at 4°C. Protein concentrations were quantified by Bicinchoninic acid (BCA) Protein Assay Kit (catalog # 23227, Thermofisher Scientific) The protein samples were separated on 10% SDS-polyacrylamide gels and electrophoretically transferred onto PVDF membranes. Then the membrane was blocked in 3% non-fat milk in phosphate-buffered saline with Tween (PBST, 0.1% Tween 20) and incubated overnight at 4°C with anti-TIMP-1 primary antibody (1:500 dilution, catalog #A00561, Boster, Wuhan, China) and anti-β-actin antibody (1:2000 dilution, catalog #4695, Santa Cruz, CA, US), followed with secondary antibody incubation at room temperature for 2 h. Blots were developed using immobilon forte western horseradish peroxidase (HRP) substrate (WBLUF0100, Millipore) and then visualized using an enhanced chemiluminescence (ECL) reagent (KeyGen Biotechnology, Nanjing, China) and detected via ECL detection system (Tanon5200, ShangHai, China). The relative levels of immunoreactive proteins were quantified using the ImageJ software.
3
B16 Melanoma Protein Profiling
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B16 melanoma cells were washed with ice-cold Phosphate buffer saline (PBS) and lysed with NP40 lysis buffer supplemented with protease inhibitor cocktail. Cells were incubated with NP40 for 30 minutes on ice. The soluble fractions of cell lysates were isolated by centrifugation at 13,000 rpm in a refrigerated microcentrifuge for 30 minutes. The protein concentration in the soluble fraction was quantified using the bicinchoninic acid (BCA) protein estimation kit.
Known concentrations of bovine serum albumin (BSA) was used to plot the standard curve.
30-50 μg of the protein was boiled in SDS dye and separated on 10% SDS PAGE gel.
Tyrosinase antibody is synthesized from Genescript. DCT (ab74073), PMEL17 (ab137078), MITF (ab12039), FASN (ab22759) and mCHERRY (ab167453) antibodies are procured from Abcam, CDK2 (MA1-81135) is obtained from Thermo Scientific while Srebf1 (04-469) is ordered from Millipore. HRP-conjugated Actin (ab8227) and Tubulin (ab6046) are used as a loading control. Horseradish peroxidase-conjugate Anti-Mouse (NA931) and Anti-Rabbit (NA934) antibodies are obtained from GE healthcare. For Western blot standard enhanced chemiluminescence reagents (WBLUF0100) were used from Millipore. ImageJ software was used for quantification.
Known concentrations of bovine serum albumin (BSA) was used to plot the standard curve.
30-50 μg of the protein was boiled in SDS dye and separated on 10% SDS PAGE gel.
Tyrosinase antibody is synthesized from Genescript. DCT (ab74073), PMEL17 (ab137078), MITF (ab12039), FASN (ab22759) and mCHERRY (ab167453) antibodies are procured from Abcam, CDK2 (MA1-81135) is obtained from Thermo Scientific while Srebf1 (04-469) is ordered from Millipore. HRP-conjugated Actin (ab8227) and Tubulin (ab6046) are used as a loading control. Horseradish peroxidase-conjugate Anti-Mouse (NA931) and Anti-Rabbit (NA934) antibodies are obtained from GE healthcare. For Western blot standard enhanced chemiluminescence reagents (WBLUF0100) were used from Millipore. ImageJ software was used for quantification.
4
Western Blot Analysis of GATA2 Protein
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10 μg of protein extracts were separated in a SDS-PAGE gel, and transferred to PVDF membranes overnight. Membranes were incubated overnight with anti-human GATA2 (clone 3C10.1, Merck Milipore) and detected by a HRP-conjugated anti-mouse IgG secondary antibody and enhanced chemiluminescent (ECL) reagents (WBLUF0100, Merck Millipore). Beta-actin or GAPDH was used as a loading control.
5
Western Blot Analysis of Protein Levels
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Liver or aortic tissue lysates were extracted in RIPA lysis buffer and protein content was measured using the DC Protein Assay Kit (Bio Rad, Hercules, CA, USA). Protein extracts (10 μg) were resolved on SDS-PAGE (10% wt/vol) and transferred electrophoretically to PVDF membranes as described previously.27 (link),32 (link)
After blocking with non-dry fat milk (5% wt/vol), membranes were probed with antibodies against VASH2 (clone 1760; provided by Tohoku University, Sendai, Japan),33 (link),34 (link)
p53 (Abcam, Cat. no. ab131442), or p21 (Abcam, Cat. no. b109199), and β-actin (Sigma-Aldrich, Cat. no. A5441). Membranes were then incubated with appropriate secondary antibodies, and immune complexes were visualized on chemiluminescence (EMD Millipore, Cat. no. WBLUF0100, Cat. no. WBLUC0100) and quantified using a General Electric Imager (LAS 4000 mini).
After blocking with non-dry fat milk (5% wt/vol), membranes were probed with antibodies against VASH2 (clone 1760; provided by Tohoku University, Sendai, Japan),33 (link),34 (link)
p53 (Abcam, Cat. no. ab131442), or p21 (Abcam, Cat. no. b109199), and β-actin (Sigma-Aldrich, Cat. no. A5441). Membranes were then incubated with appropriate secondary antibodies, and immune complexes were visualized on chemiluminescence (EMD Millipore, Cat. no. WBLUF0100, Cat. no. WBLUC0100) and quantified using a General Electric Imager (LAS 4000 mini).
6
Protein Extraction and Western Blot Analysis
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Total protein extract was isolated from human tissue using TRI Reagent (T9424, Sigma-Aldrich, Madrid, Spain) according to the manufacturer’s protocol. Total protein extracts were denatured using 1% SDS at 100 °C for 5 min. Sixteen microgram of each denatured sample was subjected to 12% SDS-PAGE and transferred to a nitrocellulose membrane (IPVH00010, Millipore, Barcelona, Spain) for 60 min at 1 mA/cm2. Membranes were incubated o.n. with primary antibodies (Ref. and dilution defined in Table 1 ) in immunoblot buffer (TBS-T-5%M; Tris-buffered saline (TBS) containing 0.05% Tween-20 and 5% no-fat dry milk) at + 4 °C in agitation and 1 h at RT upon a tilt table with HRP secondary antibodies in TBS-T-5%M. For load control, anti-alpha actin (1:20,000) was incubated for 20 min at RT in TBS-T-5%. Membrane development was performed in TBS-T-5% BSA. Membranes were developed using the Luminata Classico or Forte Western HRP Substrate (WBLUC0100 and WBLUF0100, respectively, MERCK Millipore, Darmstadt, Germany) in Fuji Medical X-Ray Films (Super RX-N; Fujifilm Co., Tokyo, Japan).
7
Western Blot Analysis of 2D and 3D Cell Lysates
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2D cultured cells were lysed in Laemmli buffer. 3D cultured primary mouse mammary cells were released from Matrigel using the BD cell recovery solution (Corning/Thermo Fisher Scientific) prior to lysis. Protein extracts were separated by SDS-PAGE on 4-15% gradient Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad), transferred to PVDF membranes (IPVH00010, Merck Millipore) and immunoblotted with anti-LYN antibodies. GAPDH was used as the loading control. The resulting immunocomplexes were detected by HRP-conjugated anti-mouse IgG or anti-rabbit IgG secondary antibodies and enhanced chemiluminescent (ECL) reagents (WBLUF0100, Merck Millipore).
8
Western Blot Analysis of Macrophage Proteins
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Whole cell proteins were extracted from macrophages using a lysis buffer (Cell Signaling, Cat. No. 9803). Each sample was applied into 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane, immunoblotted with primary antibodies (the phosphorylation of extracellular signal-regulated kinase (p-ERK); Cell Signaling, Cat. No. 9101, the phosphorylation of c-Jun N-terminal kinase (p-JNK); Cell Signaling, Cat. No. 9251, the urokinase plasminogen activator (uPA); Abcam, Cat. No. ab20789, the urokinase plasminogen activator and receptor (uPAR); R&D, Cat. No. AF534, and β-actin; Sigma Cat. No. A5441) [28 (link)]. Membranes were then incubated with appropriate secondary antibodies, and immune complexes were visualized on chemiluminescence (Merck Millipore, Cat. no. WBLUF0100, Cat. no. WBLUC0100) and quantified using a General Electric Imager (GE Healthcare, LAS 4000 mini) [29 (link)].
9
Phospho-Kinase Profiling of Mammary Cells
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3D cultured primary mammary cells were released from Matrigel using the BD cell recovery solution and lysed in Laemmli buffer. Protein extracts were separated by SDS-PAGE, transferred to PVDF membranes (IPVH00010, Merck Millipore, Hertfordshire, UK) and immunoblotted with antibodies detailed in the Key Resources Table . GAPDH or alpha-tubulin were used as loading controls. Resulting immunocomplexes were detected by HRP-conjugated anti-mouse IgG or anti-rabbit IgG secondary antibodies and enhanced chemiluminescent (ECL) reagents (WBLUF0100, Merck Millipore). Protein extracts (400 ug) from Ctr, BRCA1-, siCtr- and siPin1-MDA-MB-468 cells were processed and analyzed for phosphorylation of LYN (Y397) and SRC (Y419) using the Human Phospho-Kinase Antibody Array (R&D Systems) following the manufacturer’s instructions.
10
Analyzing Kinase Phosphorylation in 3D Cultured Mammary Cells
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