Dot-blotting was performed as previously described [29 (link)]. Briefly, aliquots of AGFC (50 μL) and phenyl Sepharose (100 μL) fractions were spotted on nitrocellulose membranes and denatured with 6 M guanidinium chloride (Merck). Membranes were blocked with low-fat dry milk (3% w/v) and incubated sequentially with anti-MATα1 antisera (1:10,000 v/v) prepared in our laboratory [32 (link)] and a secondary anti-rabbit-HRP antibody (1:10,000 v/v; BioRad, Hercules, CA, USA). The protein signal was then visualized with Western LightningTM chemiluminescence reagent (Perkin Elmer, Waltham, MA, USA). Quantification of dot-blots was performed by densitometric scanning of the images using ImageJ v1.37 software, and values were corrected for differences in fraction volumes.
Western lightning chemiluminescence reagent
Manufactured by PerkinElmer
Sourced in United States
The Western Lightning Chemiluminescence Reagent is a solution used in western blot analysis to detect and quantify specific proteins. It produces a luminescent signal that can be detected and measured using specialized imaging equipment.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (7 mentions)
Cell signaling lysis buffer, by Cell Signaling Technology (5 mentions)
β actin, by Merck Group (5 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Novex 4 12 bis tris gel, by Thermo Fisher Scientific (5 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Protease inhibitor, by Roche (3 mentions)
A5441, by Merck Group (3 mentions)
Bradford reagent, by Bio-Rad (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (3 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
Dc protein assay kit, by Bio-Rad (2 mentions)
X ray film, by Kodak (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Southern Biotech (2 mentions)
Shmt2, by Cell Signaling Technology (2 mentions)
Vinculin, by Cell Signaling Technology (2 mentions)
Shmt1, by Cell Signaling Technology (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
70 protocols using western lightning chemiluminescence reagent
1
Dot-blotting Quantification of MATα1 Protein
2
Evaluating Podocyte Endoplasmic Reticulum Stress
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After transfection for 48 h, the podocytes from each group were lysed by whole-cell lysate for 10 min on ice. The radioimmunoprecipitation assay lysis buffer was used to extract total proteins from the cultured podocytes, and a bicinchoninic acid kit was utilized to determine their concentration. The total proteins were then isolated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. After blocking in 5% skim milk at room temperature for 1 h, the membranes were probed with primary antibodies [glucose-regulated protein 78 (GRP78), 1:1000, Abcam, United States; C/EBP homologous protein (CHOP), 1:500, Abcam, United States; caspase-12, 1:1000, Abcam, United States] overnight at 4 °C and then incubated with the secondary antibody for 1 h. The membranes were washed three times with PBST for 10 min, incubated in Western LightningTM Chemiluminescence Reagent (PerkinElmer, United States) for 5 min, and visualized by the LabWorksTM imaging system. β-Tubulin (1:1000, Abcam, United States) was used as an internal control. ImageJ software (National Institutes of Health, Bethesda, MD, United States) was used to analyze the gray value of the target band.
3
Western Blot Analysis Protocol
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Western LightningTM Chemiluminescence Reagent was obtained from PerkinElmer, USA (lot NEL103 001EA); goat anti-rabbit IgG secondary antibody was supplied by Abcam, UK (lot ab6721); rabbit anti-mouse IgG secondary antibody was supplied by Abcam, UK (lot ab6721ab6728); M-mlv reverse transcriptase was obtained from Takara, Japan (lot 2641A); RNase inhibitors were purchased from Takara, Japan (lot D2310 C); SYBR Premix Ex Taq was obtained from Takara, Japan (lot DRR041 A); Hq-350xt development and fixing equipment was supplied by Suzhou Huqiu Image Equipment Co., Ltd., China; Superrx film was supplied by FUJIFILM, Japan; Decolorizing Orbital Shaker Ts-1 was supplied by Jiangsu Haimen Qilin Bell Instrument Manufacturing Co., Ltd., China; Dycz-24dn vertical electrophoresis device was supplied by Beijing Liuyi Instrument Factory, China; Ve-386 transfer electrophoresis tank was supplied by Beijing Yuanpinghao Biotechnology Co., Ltd., China; 5415D centrifuge was supplied by Eppendorf, Germany; Bio-Rad real-time PCR amplification instrument was supplied by Bio-Rad, USA; MAKT1 antibody was obtained from Cell Signaling Technology, USA (cat.4695S); AKT1 antibody was purchased from Abcam, UK (cat. ab81283); TNF antibody was obtained from Abcam, UK (cat. ab6671); VEGFA antibody was supplied by Abcam, UK (cat.ab1316); and EGFR antibody was purchased from Abcam, UK (cat.ab52894).
4
Western Blot Analysis of Autophagy Markers
5
FAK Protein Expression Analysis
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On day 10, cells cultured on Ti surfaces with or without PF-573228 were lysed, and the protein of each cell was extracted and transferred to PVDF membrane as previously described9 (link) to evaluate FAK protein expression by Western Blotting assay. Blocking of non-specific sites was performed with 5% Non-Fat Dry Milk Blotting Grade Blocker (Bio-Rad Laboratories, Hercules, CA, USA) for 2 h. Cells were incubated overnight at 4°C with a rabbit polyclonal antibody to FAK (1:1000, Cell Signaling Technology, Denver, MA, USA) and a mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (1:2000, Santa Cruz Biotechnology), which was used as a control. Secondary antibodies conjugated to HRP (1:2000, Santa Cruz Biotechnology) were used for immunodetection with Western Lightning Chemiluminescence Reagent (PerkinElmer Life Sciences, Waltham, MA, USA), and images were captured by a G:Box gel imaging system (Syngene, Cambridge, UK).
6
MGO-Induced Oxidative Stress in HUVECs
7
Immunoblotting of Dephosphorylated IRE1
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Cells were lysed in radioimmunoprecipitation assay buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 1 mM EDTA) supplemented with protease inhibitors (Roche) and phosphatase inhibitors. Protein concentrations were determined by bicinchoninic acid assays (Thermo Fisher Scientific). IRE1 was immunoprecipitated with an anti–mouse IRE1 antibody and protein G–Sepharose beads (Sigma-Aldrich). Bead-bound IRE1 was dephosphorylated using λPPase (New England Biolabs, Inc.) or CIP (New England Biolabs, Inc.). Proteins were boiled in SDS-PAGE sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, and 0.1% bromophenol blue) with β-ME, analyzed by SDS-PAGE, and transferred to nitrocellulose membranes, which were then blocked in 5% nonfat milk (wt/vol in PBS) and immunoblotted with the indicated primary antibodies and appropriate HRP-conjugated secondary antibodies. Immunoblots were developed with Western Lightning chemiluminescence reagent (Perkin-Elmer).
8
Quantitative Immunoblot Analysis of Rac1 and Cortactin
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Antibodies to cortactin (16-228) and Rac1 (05-389) were from Millipore (MA) whereas antibodies to hemagglutinin epitope (2362) and Hax-1 (H65220) were from Cell Signaling (Beverly, MA) and BD Biosciences (San Jose, CA), respectively. GAPDH antibody was from Ambion (4300). Peroxidase-conjugated anti-rabbit IgG (W401B and anti-mouse IgG (NA931V) were purchased from Promega (Madison, WI). 1mg of the lysate protein of the experimental groups were subjected to immunoprecipitation using the Rac1 or cortactin antibodies. The immunoprecipitates were resolved by 10% or 15% SDS-PAGE gels, and immunoblot analyses were carried out according to our previously published methods [5 (link), 34 (link), 49 (link)]. The blots were developed using Western Lightning Chemiluminescence Reagent (Perkin Elmer, Boston MA) and imaged using Kodak Image Station 4000 MM. Quantification of immunoblots was performed using Carestream Molecular Imaging Software version 5 (Rochester, NY) and the respective values were imported into Graph Pad Prism (La Jolla, CA) for graphing and statistical analysis.
9
Quantitative SARS-CoV-2 RBD Immunoblot Analysis
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The RBDs (0.3 μg) and IgYs (0.5 μg) were separated using SDS-PAGE and stained with Coomassie. For immunoblot analysis, 0.3 μg RBD was separated using SDS-PAGE and transferred to a nitrocellulose membrane (Sartorius). The membranes were blocked with 10% BSA (Santa Cruz Biotechnology) in TBS. Primary IgY antibodies were diluted in TBS (5 μg/mL) and added to the membranes. After 2 h incubation at room temperature, membranes were washed three times with TBS buffer containing 0.03% Tween 20 (Tween/TBS). Secondary antibody conjugated to HRP (1:5000, goat-anti-chicken IgY, GtxCk-003-DHRPX, ImmunoReagents) was added. After 1 h, chemiluminescence was detected using the Western Lightning Chemiluminescence Reagent (Perkin Elmer) by the ChemiDoc Imaging System (BioRad).
10
BCoV Nucleocapsid Protein Detection
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BHK-21 cells in 35-mm dishes at ~80 % confluency (~8 × 105 cells/dish) were infected with BCoV at an MOI of 1 PFU/cell. Cell lysates were harvested at different time points of infection, electrophoresed through 12 % SDS-PAGE gels, and electrotransferred to nitrocellulose membranes (Amersham Biosciences). BCoV nucleocapsid (N) proteins were detected using an antibody specific to the BCoV N protein as the primary antibody and goat anti-mouse IgG conjugated to HRPO as the secondary antibody (Jackson Laboratory). The proteins detected were visualized using Western Lightning™ Chemiluminescence Reagent (Perkin Elmer NEL105) and X-ray film (Kodak).
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