Cultured cells in an exponentially growing phase and frozen tissue samples were extracted with 1 ml of lysis buffer. The protein concentration of the lysate was determined by the BCA method (KeyGen, China). The extracts containing approximately 30 μg of protein were loaded onto 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), electrophoresed, and then transferred to polyvinylidence difluoride (PVDF) membranes (Immobilon-P. 0.45 μm, Millipore Corp., Bedford, MA, USA). The membranes were blocked with 5 % skimmed milk in PBST (phosphate-buffered saline with 0.1 % Tween 20) for 1 h and then incubated with primary antibody of rabbit anti-ECM1 antibody (Protein Group Inc, USA), rabbit anti-Vimentin antibody (ab92574, Abcam, USA), rabbit anti-E-cadherin antibody (ab15148, Abcam, USA), and rabbit anti-β-actin (ab133626, Abcam, USA) antibody at 4 °C overnight. After being washed three times with PBST buffer, the membranes were incubated with secondary peroxidase-conjugated antibody (ZB-2308, ZSGB-BIO) for 1 h at room temperature. After being washed three more times, the protein bands were detected by enhanced chemiluminescence (Pierce, Rockford, USA) according to the manufacturer’s instruction. β-actin antibody was served as a control to confirm equal loading. Densitometry index analysis of the bands was made using gel imagery system.
Immobilon p 0.45 μm
Manufactured by Merck Group
Sourced in United States
Immobilon-P. 0.45 μm is a type of lab equipment used for sample preparation and analysis. It is a hydrophobic polyvinylidene difluoride (PVDF) membrane with a pore size of 0.45 micrometers. This membrane is designed for protein transfer and immobilization in common laboratory techniques such as Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Ab15148, by Abcam (1 mentions)
Ab92574, by Abcam (1 mentions)
Ab133626, by Abcam (1 mentions)
Zb 2308, by ZSGB-BIO (1 mentions)
Bca method, by Keygen Biotech (1 mentions)
Image lab software version 3, by Bio-Rad (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Immobilon western, by Merck Group (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Precision plus protein standards kaleidoscope, by Bio-Rad (1 mentions)
Westernbright ecl, by Advansta (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Antibodies against histone 3, by Abcam (1 mentions)
Rabbit anti nf κb p65, by Cell Signaling Technology (1 mentions)
Mab360, by Merck Group (1 mentions)
Mab3418, by Merck Group (1 mentions)
Mouse anti iκb, by Cell Signaling Technology (1 mentions)
Ab15191, by Abcam (1 mentions)
Mouse anti phospho iκb, by Cell Signaling Technology (1 mentions)
Image station 4000r, by Kodak (1 mentions)
10 protocols using immobilon p 0.45 μm
1
Western Blot Analysis of Cell Proteins
2
Whole Cell Lysate Fractionation and Western Blot Analysis
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Whole cell lysate was prepared by solubilizing cell pellet in RIPA buffer (Sigma; R-0278) over ice for 40 minutes as described previously [32 (link)]. The lysate was cleared by centrifugation at 14000 g for 15 min at 4°C and the supernatant was collected as whole cell lysate. Cytosolic and nuclear fractions were prepared as described previously with minor modifications [33 (link)]. Protein estimation was done by Bradford’s method.
Western blotting was performed using equal amounts of protein, including pre-stained molecular weight markers (Precision Plus Protein Standards Kaleidoscope, Bio Rad). The proteins were electrotransferred to polyvinylidene difluoride (PVDF) membranes (Immobilon P 0.45 μm, Millipore). After blocking for 2 h at room temperature in 5% fat-free milk powder or 3% BSA in TBST (Tris-buffered saline containing 0.05% Tween 20, pH 8.0), membranes were incubated overnight with specific antibodies. After 30 minutes washing with TBST, membranes were probed with different HRP-conjugated secondary antibodies. Detection of specific proteins was carried out by Immobilon Western (Chemiluminescent HRP substrate, Millipore) mediated chemiluminescence by ChemiDoc XRS+ (Bio Rad). Densitometry of specific bands was done using Image Lab Software version 3 (Bio Rad).
Western blotting was performed using equal amounts of protein, including pre-stained molecular weight markers (Precision Plus Protein Standards Kaleidoscope, Bio Rad). The proteins were electrotransferred to polyvinylidene difluoride (PVDF) membranes (Immobilon P 0.45 μm, Millipore). After blocking for 2 h at room temperature in 5% fat-free milk powder or 3% BSA in TBST (Tris-buffered saline containing 0.05% Tween 20, pH 8.0), membranes were incubated overnight with specific antibodies. After 30 minutes washing with TBST, membranes were probed with different HRP-conjugated secondary antibodies. Detection of specific proteins was carried out by Immobilon Western (Chemiluminescent HRP substrate, Millipore) mediated chemiluminescence by ChemiDoc XRS+ (Bio Rad). Densitometry of specific bands was done using Image Lab Software version 3 (Bio Rad).
3
Immunoblotting Protocol for Protein Detection
4
Western Blot Analysis of Inflammatory Markers
5
Immunoblotting Analysis of SLC26A3 and SLC26A6
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Electrophoresis of protein samples was conducted using 4–20% gradient Tris-HEPES-SDS precast mini gels (Thermo Scientific, Rockford, IL). After electrophoresis, gels were soaked three times, 10 min each, in transfer buffer (192 mmol/L glycine, 25 mmol/L Tris base, and 10% methanol) to remove SDS and to prevent gel shrinkage. Proteins were transferred to polyvinylidene difluoride membranes (Immobilon-P 0.45 μm, Millipore, Bedford, MA) for 4 h under 2 mA/cm2 at 4°C. After transfer, membranes were blocked with 5% dry milk in PBS overnight. Membranes were then incubated with primary antibody for 12 h at 4°C. After washing with PBS-Tween buffer (0.1% Tween 20 in PBS, pH 7.5; five washes, 5 min, each), membranes were exposed to a suitable horseradish peroxidase (HRP)-conjugated secondary antibody (Thermo Scientific). Membranes were then incubated with SuperSignal West Femto Chemiluminescent Substrate (Pierce). Gel images were captured digitally (Imagestation 4000R, Eastman Kodak Co., Rochester, NY). To detect SLC26A3 or SLC26A6 on immunoblots, 1 μg/mL of primary antibody and 10 ng/mL of HRP-conjugated IgG were employed.
6
Analyzing Titin Post-Translational Modifications
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To analyze titin post-translational modification, LV tissues samples were homogenized in modified Laemmli buffer. Samples were heated at 96 °C for 3 min, centrifuged for 3 min at 4 °C at 14,000 rpm, and then separated via agarose strengthened 2% SDS-PAGE [7 (link),67 (link)]. Gels were run at 2–4 mA constant current per gel for 16 h. Proteins were blotted on PVDF membranes (Immobilon-P 0.45 μm; Merck Millipore, Burlington, MA, USA). Blots were pre-incubated with 5% BSA in TBST for 1 h at RT followed by primary antibody incubation overnight at 4 °C. Titin ubiquitination was determined via an anti-ubiquitin antibody (Table 1 ) and glutathionylation of titin via an anti-GSH antibody (Table 1 ). After washing with TBST, primary antibodies were detected with HRP-conjugated secondary anti-rabbit or anti-mouse antibodies (Table 2 ) and enhanced chemiluminescence (Clarity Western ECL Substrate, BioRad, Munich, Germany). Chemiluminescence signals were normalized to signals obtained from Coomassie-stained PVDF membranes referring to the entire protein amount transferred. The results were quantitated via densitometry using Multi Gauge V3.2 software.
7
Evaluating Cell Death Pathways by GNSbM Extract
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To determine the induced cell death pathway, 20, 30, and 50 μg/mL of GNSbM extract were exposed on both cell lines for 48 h. Additionally, as positive controls: cells were exposed for 10 min to UV radiation (Osram, G30T8, 30W Germicidal UV-C Lamp, 254 nm) for apoptosis induction [13 (link)] or for 1 h to PBS for starvation-induced autophagy [14 (link)], before being harvested. Apoptotic and autophagic proteins were analyzed through Western Blot analysis. The methodology was applied according to Bailon-Moscoso et al. [15 (link)]. Briefly, separated proteins from a SDS-PAGE were transferred to a PVDF membrane (IPVH00010, Immobilon-P, 0.45 μm, EMD/Millipore, Billerica, Boston, MA, USA) and incubated with primary antibodies: p53 (sc-81168), Beclin-1 (sc-48341), SQSTM 1/p62 (sc-48402) (Santa Cruz Biotechnology, USA), PARP (#9542), Bax (#2774), Bcl-2 (#15071) LC3A/B (#12741), and β-Tubulin (#2128) (Cell Signaling Technology, USA), as indicated by the manufacturer for immunoblotting. Secondary antibodies, anti-mouse IgG, HRP-linked (#7076, Cell Signaling Technology, USA), and anti-rabbit IgG, HRP-linked (#7074, Cell Signaling Technology, USA), were subsequently used. Immunoreactive bands were visualized using an enhanced chemiluminescence Luminata™ Crescendo Western HRP Substrate or Luminata™ Forte Western HRP Substrate (Millipore-Merck, Germany).
8
Detecting Titin Ubiquitination and Oxidation
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To detect titin ubiquitination, LV samples were solubilized in a modified Laemmli buffer (50 mM Tris-HCl at pH 6.8, 8 M urea, 2 M thiourea, 3% SDS w/v, 0.03% ServaBlue w/v, 10% v/v glycerol, 75 mM DTT). For detecting titin oxidation, N-ethylmaleimide instead of DTT was used for solubilization. Samples were heated at 96 °C for 3 min, centrifuged for 3 min at 4 °C at 14,000 rpm, and then separated by agarose-strengthened 2% SDS-PAGE [28 (link),29 (link)]. Gels were run at 2–4 mA constant current per gel for 16 h. Following SDS-PAGE, proteins were blotted onto PVDF membranes (Immobilon-P 0.45 μm; Merck Millipore, Burlington, MA, USA). Blots were preincubated with 4% bovine serum albumin in TBST for 1 h at RT followed by primary antibody incubation overnight at 4 °C.
Titin ubiquitination was investigated by HRP-conjugated secondary antirabbit antibodies (1:10,000), which were added the next day for 1 h at RT. After washing steps, blots were treated with ECL for developing chemiluminescence signal (see above). Chemiluminescence signals were normalized to signals obtained from Coomassie-stained PVDF membranes regarding the total amount of protein transferred. The results were quantified by densitometry using Multi Gauge V3.2 software (Fujifilm Ltd, Tokyo, Kanto, Japan).
Titin ubiquitination was investigated by HRP-conjugated secondary antirabbit antibodies (1:10,000), which were added the next day for 1 h at RT. After washing steps, blots were treated with ECL for developing chemiluminescence signal (see above). Chemiluminescence signals were normalized to signals obtained from Coomassie-stained PVDF membranes regarding the total amount of protein transferred. The results were quantified by densitometry using Multi Gauge V3.2 software (Fujifilm Ltd, Tokyo, Kanto, Japan).
9
Western Blot Analysis of AMPK Signaling
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Sepasol-RNA I Super G was purchased from Nacalai Tesque (Kyoto, Japan). Isopentane, formaldehyde, 2-mercaptoethanol, and 1% eosin Y solution were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). The PrimeScript RT Reagent Kit with gDNA Eraser was purchased from TaKaRa Bio (Shiga, Japan). SYBR FAST qPCR kits were purchased from Kapa Biosystems (Wilmington, MA, USA). Primary antibodies against AMPKα (#2532), phosphorylated Thr-172 AMPKα (#2535), Akt (#9272), phosphorylated Ser-473 Akt (#4058), forkhead box protein O1 (FOXO1) (#2880), phosphorylated Ser-256 FOXO1 (#9461), FOXO3a (#12829), and phosphorylated Ser-253 FOXO3a (#13129) were purchased from Cell Signaling Technology (Danvers, MA, USA); MEF2A (sc-313), PGC-1α (sc-517380), and Sp1 (sc-14027) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA); and α-tubulin (#017-25031) was obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). For secondary antibodies, goat anti-mouse IgG H&L (HRP) (ab6789) and goat anti-rabbit IgG H&L (HRP) (ab6721) were purchased from Abcam plc. (Cambridge, UK), and goat anti-rabbit IgG-HRP (sc-2004) was purchased from Santa Cruz Biotechnology. Polyvinylidene difluoride membrane (Immobilon-P, 0.45μm) for Western blotting was purchased from Merck (Darmstadt, Germany).
10
SDS-PAGE and Immunoblotting Analysis
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Equal volumes of the harvested proteins were subjected to SDS-PAGE using 10% polyacrylamide gel and separated electrophoretically with a Tris-glycine buffer (25 mM Tris, 0.192 M glycine, 0.1% SDS). Gels were then subjected to Coomassie Brilliant Blue (CBB) staining or immunoblotting. For immunoblotting, separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Immobilon-P 0.45 μm, Merck Millipore) with a semi-dry blotting apparatus (ATTO Corporation, Tokyo, Japan) at 240 mA for 2 h at 4°C using buffer containing 192 mM glycine, 100 mM Tris and 5% methanol. In some experiments, the protein spots on the 2D-PAGE gels were also electro-blotted onto a PVDF membrane as described above. The membrane was then blocked with 0.5% (w/v) skim milk in PBS containing 0.1% (w/v) Tween-20 for 1 h at room temperature. The membrane was incubated with primary antibodies, calreticulin (1:500), PSD-95 (1:1000), GM130 (1:200) or TfR (1:1000), overnight at 4°C. The membrane was then treated with HRP-conjugated anti-mouse or rabbit IgG for 1 h at room temperature. Immunoreactive spots were detected using an enhanced chemiluminescence detection kit (ECL plus, GE Healthcare).
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