The cellular total protein were obtained by a protein extraction kit. Besides, the Bradford protein assay kit (BESTBIO) was used to determine the protein concentration. 30 μg or 50 μg of total cell extracts were analyzed by Western blotting. The proteins were loaded onto 15% SDS-PAGE gels and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). Membranes were incubated with 1 μg/ml Rabbit anti-ATG4a antibody (Polyclonal, ABGENT), 2 μg/ml mouse anti-SQSTM1/p62 antibody (Monoclonal, Abcam), 2 μg/ml rabbit anti-LC3 antibody (Cell Signaling Technology, Inc.), 2.5 μg/ml rabbit anti-GAPDH antibody (Monoclonal, Cell Signaling Technology, Inc.) or 2 μg/ml mouse anti-β-actin antibody (Monoclonal, Cell Signaling Technology, Inc.). After that, the membranes were incubated with 1 μg/ml HRP-conjugated goat anti-rabbit IgG secondary antibody or 1 μg/ml HRP-conjugated goat anti-mouse IgG secondary antibody (Proteintech Group, Inc.). Immunoreactive band analysis was performed by using the enhanced western bright ECL reagent (Advansta, United States). Densitometry analyses of western blots were performed with ImageJ software (version 1.46). Western blots were developed to be linear in the range used for densitometry. All results were expressed as a relative ratio to the β-actin or GAPHD. At least three or four independent western blot experiments were performed.
Enhanced western bright ecl reagent
Manufactured by Advansta
Sourced in United States
Enhanced Western Bright ECL reagent is a chemiluminescent substrate for the detection of horseradish peroxidase (HRP) in Western blot analysis. It provides a bright, stable signal for sensitive detection of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Bradford protein assay kit, by BestBio (2 mentions)
Protein assay, by Bio-Rad (2 mentions)
Mouse anti β actin antibody, by Cell Signaling Technology (1 mentions)
Hrp conjugated goat anti mouse igg secondary antibody, by Proteintech (1 mentions)
Rabbit anti gapdh antibody, by Cell Signaling Technology (1 mentions)
Rabbit anti lc3 antibody, by Cell Signaling Technology (1 mentions)
Mouse anti sqstm1 p62 antibody, by Abcam (1 mentions)
Hrp conjugated goat anti rabbit igg secondary antibody, by Proteintech (1 mentions)
4 protocols using enhanced western bright ecl reagent
1
Quantitative Western Blot Analysis of Autophagy Markers
2
Western Blot Analysis of Autophagy Markers
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To obtain total cellular lysates, the cells were lysed in an ice-cold cell lysis buffer from a protein extraction kit. The protein concentration was determined with the Bradford protein assay kit (BESTBIO) using a c-globulin standard curve. Proteins were resolved by standard SDS-PAGE and transferred onto PVDF membranes (Millipore, USA). Nonspecific binding sites were blocked using 5% dry skimmed milk, 0.2% Tween-20 in PBS (pH 7.4) for overnight at 4°C and then incubated with primary antibodies againstULK1、 LC3I/II and GAPDH (Cell Signaling Technology, Inc, Danvers, MA, USA). The membranes were then incubated at room temperature (RT) for 1 h with appropriate HRP-conjugated secondary antibodies prior to be developed using the enhanced Western Bright ECL reagent (Advansta, United States).
3
Western Blot Analysis of Protein Signaling
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Whole cell extracts were prepared by homogenizing cells in lysis buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 0.5% NP-40) for 60 min on ice. The lysates were then centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatants were collected for analysis. The soluble protein concentration was measured with Bio-Rad Protein Assay (Bio-Rad Laboratories, Richmond, CA) using bovine serum albumin (BSA) to generate a standard curve. The final clarified lysates (100 µg) were separated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel (SDS-PAGE) and transferred to nitrocellulose membranes for Western blotting assay. The membrane was blocked in 5% fat-free dry milk in PBS containing 0.3% Tween-20, and probed with antibodies against GAPDH, TLR6, NFκB, TRAF6 and/or MyD88 (All antibodies were products of Santa Cruz Biotechnology, United States). The blots were then developed using the enhanced Western Bright ECL reagent (Advansta, United States).
4
Whole Cell Protein Extraction and Immunoblotting
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Whole cell extracts were prepared by lysing cell cultures in lysis buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 0.5% NP-40) for 60 min on ice. The concentration of soluble protein was determined with Bio-Rad Protein Assay based on the method of Bradford (Bio-Rad Laboratories, Richmond, CA, USA). The clarified lysates (100 μg) were resolved in 8% or 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel (SDS-PAGE) and then transferred to nitrocellulose membranes for immunoblotting assay probed with antibodies to proteins of interest. The primary antibodies used in this study were listed in Suppl. Table 2 . All these primary antibodies were applied in a dilution of 1:500–1000. Following extensive washing, protein of interest was detected or visualized with an appropriate HRP-labelled or fluorescence-labelled IRDye (Li-Cor Biosciences, Lincoln, NE, USA) secondary antibody. The blots were then developed using the enhanced Western Bright ECL reagent (Advansta, Menlo Park, CA, United States) or Li-Cor Odassay Scanner (Li-Cor Biosciences). The relative expression of protein was semi-quantified by optical densitometry using ImageJ Software version 1.46 (http://rsb.info.nih.gov/ij/ ). The densitometric arbitrary unit (A.U.) was used for determined the ratio between the net intensity by calculating values of each sample divided by the β-actin internal control.
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