To obtain total cell lysate, cells were collected by trypsinization, washed twice with ice‐cold 1× PBS and lysed for 1 h on ice by adding cell lysis buffer. Protein concentration was measured using the BCA Protein Assay kit according to the manufacturer's instructions. Total lysates were loaded on SDS‐PAGE, and proteins were separated using SDS running buffer. A PageRuler prestained protein ladder was loaded on the same gel as a standard for protein separation and determination of molecular weight. Proteins were transferred onto polyvinylidene difluoride (PVDF) membranes. After blocking, membranes were incubated overnight with primary antibodies against PAX2 (1 : 400, Santa Cruz, USA), CD55 (1 : 100, Santa Cruz, USA), C3 (1 : 100, Santa Cruz, USA), CFH (1 : 200, Bioss antibodies, China). Subsequently, membranes were washed three times in 1× TBST for 10 min, then incubated with corresponding horseradish peroxidase (HRP)‐conjugated isotype‐specific secondary antibody diluted in 5% non‐fat milk, for 1 h at room temperature. After four washes with 1× TBST, blots were visualized with an ECL + plus™ western blotting system (Amersham, USA) and films. GAPDH (1 : 5000, Bioss antibodies, China) was used as an internal control. The image was analyzed with the quantity ONE system.
Ecl plus western blotting system
Manufactured by Cytiva
Sourced in United States, United Kingdom
The ECL Plus Western Blotting System is a laboratory equipment designed for the detection and analysis of proteins in western blotting procedures. It provides a chemiluminescent detection method for visualizing protein bands on a membrane.
Automatically generated - may contain errors
Lab products found in correlation
Mouse α β actin, by Merck Group (2 mentions)
Gapdh, by Bioss Antibodies (1 mentions)
Polyvinylidene fluoride (pvdf), by Cytiva (1 mentions)
Rabbit α eif4e, by Cell Signaling Technology (1 mentions)
Rabbit α parp, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Anti ccnb1, by Proteintech (1 mentions)
Anti β actin, by Abcam (1 mentions)
Horseradish peroxidase, by Cell Signaling Technology (1 mentions)
Anti rabbit igg secondary antibody, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
P mtor ser2448, by Cell Signaling Technology (1 mentions)
P ampk, by Cell Signaling Technology (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Bcl xl, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
E cadherin, by BD (1 mentions)
Anti gapdh antibody, by Beyotime (1 mentions)
10 protocols using ecl plus western blotting system
1
Western Blot Analysis of Complement Proteins
2
Western Blot Analysis of Cap-Binding Proteins
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Protein samples for the cap-affinity assay were separated by 8-15% gradient SDS-PAGE (polyacrylamide gel electrophoresis). Ten or twelve percent SDS-PAGE was used for PARP and eIF4E proteins, respectively. Following protein transfer to PVDF (Amersham Biosciences) the membranes were blocked in 5% non-fat dry milk for 1 hour at room temperature in Tris-buffered saline-Tween (TBST: 0.15 M NaCl; 0.01 M Tris-HCl, pH 7.6; 0.05% Tween 20). The membranes were then incubated for 1 hour at ambient temperature or overnight at 4°C with the chosen primary antibody. The primary antibodies employed were rabbit α-eIF4E (catalog number 9742), rabbit α-PARP (catalog number 9542), α-SV40 large T antigen (catalog number 15729) all from Cell Signaling Technology (Danvers, MA) at a 1:1000 dilution, mouse α-β-actin (Sigma-Aldrich, catalog number A1978) at a 1:10,000 dilution and rabbit α-eIF4GI (kindly provided by Nahum Sonenberg, McGill University Montreal, QC, Canada) at a 1:2500 dilution. Preceding and following incubation with the appropriate horseradish peroxidase labeled secondary antibodies, the blots were washed three times for 5 minutes in TBST and detection was then performed utilizing ECL Plus Western Blotting System (Amersham Biosciences) to visualize the bands of interest.
3
Western Blot Analysis of FAP, β-actin and CCNB1
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Protein extracts from cells were prepared in RIPA buffer (10 mM Tris, pH 7.5, 0.1 M NaCl, 1 mM EDTA, and 0.1% Triton X-100). The protein was subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose membranes (Millipore). The membranes were blocked with TBST buffer (20 mM Tris, pH 7.5, 500 mM NaCl, 0.05% Tween-20, and 0.2% Triton X-100) containing 5% skimmed milk and then incubated with the primary antibodies against different proteins at 4 °C overnight, followed by incubation with a secondary antibody. The bands were visualized using the ECL Plus Western blotting system (Amersham). The antibodies used in Western blot analysis included anti-FAP (AF3715, RD), anti-β-actin (Abcam) and anti-CCNB1 (Proteintech).
4
Rabbit Immunization for Histone Antibodies
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Randomly bred female New Zealand white rabbits weighing 1–1.5 Kg were selected for immunizationas per the established protocol of our lab [27 (link)]. Serum immunoglobulin G (IgG)from the experimental animals was isolated by affinity chromatography on protein A-agarose column [28 (link)] and Western blotting was performed as per the protocol given in the technical booklet for ECL Plus Western Blotting system (Amersham, UK) to establish the specificity of antibodies generated against native and modified histones H1 in the rabbits [29 ].
5
Western Blot Analysis of PG-1 Protein
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Culture supernatants were subjected to 12% SDS-PAGE and electrophorectically transferred to 0.45 μm pore size PVDF membrane (Millipore Inc., Burlington, CA, United States). The membrane was blocked in 5% (w/v) skim milk in TBST buffer (0.3% Tris, 0.8% NaCl, 0.02% KCl, 0.1% Tween 20) for 1 h and then incubated with affinity purified polyclonal rabbit anti-PG-1 antibody at 1:500 dilution, synthesized from Biomatik (Cambridge, ON, Canada) overnight at 4°C. After washing the membrane three times at 5 min each in TBST, the membrane was incubated with anti-rabbit IgG secondary antibody (1:1000 dilution) conjugated to horseradish peroxidase (Cell Signaling, Danvers, MA, United States) for 1 h at room temperature. Bands were visualized using an ECL Plus Western blotting system according to manufacturer’s instruction (Amersham Biosciences, Piscataway, NJ, United States).
6
Protein Expression Analysis by Western Blot
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Protein samples were separated by 10% SDS-PAGE (polyacrylamide gel electrophoresis) or by 8–15% gradient gels. Following protein transfer to PVDF the membranes were blocked in 5% non-fat dry milk for 1 hour at room temperature in Tris-buffered saline-Tween (TBST: 0.15 M NaCl; 0.01 M Tris-HCl, pH 7.6; 0.05% Tween 20). The membranes were then incubated for 1 hour at ambient temperature or overnight at 4°C with the chosen primary antibody. The primary antibodies employed from Cell Signaling were rabbit α-STAT1 [#9172], rabbit α-Phospho-STAT1 (Tyr701) antibody [#9171], mouse α-STAT3 [#9139], rabbit α-Phospho-STAT3 (Tyr705) antibody [#9145], rabbit α-PKR antibody, rabbit α-PERK antibody [#3192], rabbit α-Ero1-Lα antibody [#3264], mouse α-CHOP antibody [#2895], rabbit α-Calnexin antibody [#2679], rabbit α-IRE1α antibody [#3192] and rabbit α-BiP antibody [#3177] each at a 1:1000 dilution. Other primary antibodies utilized were rabbit α-p48 antibody [sc-496] from Santa Cruz Biotechnology at 1:500 dilution and mouse α-β-actin [A1978] (Sigma) at a 1:10,000 dilution. Preceding and following incubation with the appropriate horseradish peroxidase labeled secondary antibody, the blots were washed three times for 5 minutes in TBST. Detection was performed utilizing ECL Plus Western Blotting System (Amersham Biosciences) to visualize the bands of interest.
7
Western Blot Analysis of Protein Samples
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Protein samples were separated by 10% SDS-PAGE (polyacrylamide gel electrophoresis) or by 8-15% gradient gels. After protein transfer to PVDF (polyvinylidene difluoride), the membranes were blocked in 5% non-fat dry milk for 1 hour at room temperature in Tris-buffered saline-Tween (TBST: 0.15 M NaCl; 0.01 M Tris-HCl, pH 7.6; 0.05% Tween 20). Membranes were then incubated for 1 hour at ambient temperature or overnight at 4o C with the chosen primary antibody. The primary antibodies employed were rabbit α-HSP70 antibody (Cell Signaling), rabbit α-PARP antibody (Cell Signaling), each at a 1:1000 dilution, and mouse α-β-actin (Sigma) at 1:10,000 dilution. Before and after incubation with the appropriate horseradish peroxidase labeled secondary antibodies, the blots were washed three times for 5 minutes in TBST. Detection was performed utilizing ECL Plus Western Blotting System (Amersham Biosciences) to visualize the bands of interest. Protein band density was measured by using ImageJ, a public domain java image processing program.
8
Western Blot Analysis of Cellular Signaling
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Western blot analysis of cell and tissue lysates was performed as described previously [20] . The primary antibodies used include: p-AMPK (Thr172), AMPK, p-ACC (Ser79), ACC, p-mTOR (Ser2448), mTOR, Bax, Bcl-2, Bcl-xl, caspase 3 and cleaved caspase 3 from Cell Signaling Technology (Beverly, MA, USA); N-cadherin and actin antibodies from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and E-cadherin from BD Biosciences. After incubation with corresponding secondary antibodies, the signals were detected using ECL plus Western blotting system (Amersham, Piscataway, NJ, USA). The bands of p-AMPK and AMPK were quantified using ImageJ, and the expression level of p-AMPK relative to that of AMPK (i.e.
relative expression level) was analyzed.
relative expression level) was analyzed.
9
Western Blot Protein Detection Protocol
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Cells were lysed in NP-40 lysis buffer (Beyotime, Shanghai, China) on ice for 20 min. Protein concentrations were determined using a BCA protein assay kit (HyClone-Pierce, LA, USA). Protein samples (36 µg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto membranes (Millipore, USA). The membranes were blocked with 5% non-fat milk in Tris-buffered saline and Tween 20 (10 mM Tris-HCl, pH 8.0, 100 mM NaCl and 0.05% Tween, TBS-T). The membranes were then probed with mouse anti-NEDD9 antibody (1:2,000) (Abcam, MA, USA) or anti-GAPDH antibody (1:10,000) (Beyotime, Shanghai, China) at 4 °C overnight. Membranes were washed 3 times with TBS-T and incubated with horseradish peroxidase-conjugated goat anti-mouse secondary antibody (1:3,000) (Santa Cruz Biotechnology, USA) for 2 h at room temperature. The immunohistochemical reaction of bound antibodies was visualized using ECL PlusTM western blotting system (Amersham Pharmacia Biotech, UK).
10
Lentiviral Vector-Mediated Gene Silencing in MCF-7 and HEK293T Cells
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The human breast cancer cell line MCF-7 and the cell line HEK293T were purchased from the cell bank of the Biochemistry and Cell Biology Institutes, Shanghai Institutes for Biological Sciences (Shanghai, China). The lentiviral vector system pLVTHM, psPAX2, and pMD2G were purchased from Tronolab (www.tronolab.com/lentivectors.php) . BamHI and EcoRI restrictive endonuclease and T4 ligase were purchased from New England Biolabs (Ipswich, MA, USA). Cell culture media RPMI-1640 and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). The plasmid extract kit was purchased from Qiagen (Hilden, Germany). TRIzol reagent and Lipofectamine TM 2000 Oligofectamine reagent were purchased from Invitrogen (Carlsbad, CA, USA). M-MLV reverse transcriptase was purchased from Promega (Madison, WI, USA). SYBR Master Mixture were purchased from TaKaRa (Shiga, Japan). The BCA protein analysis kit was purchased from HyClone-Pierce (Logan, UT, USA). Rabbit anti-EMMPRIN antibody was purchased from Abcam (Cambridge, UK). Mouse anti-glyceraldehyde 3-phosphate dehydrogenase antibody, peroxidase-labeling goat anti-rabbit IgG, and goat anti-mouse IgG were purchased from Santa Cruz Biotechnology. The ECL-Plus TM Western Blotting System was purchased from Amersham-Pharmacia Biotech (Amersham, UK).
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