Infected BMDM were washed with PBS, lysed in 1x SDS-PAGE buffer on ice, and heat sterilized for 30 min at 100°C. Total protein lysates were analyzed by SDS-PAGE using pre-cast Tris-HCl criterion gels (Bio-Rad, Hercules, CA). The following primary antibodies were used: rabbit antibody against HIF-1α (NB100-479, Novus Biologicals, Littleton, CO and also D2U3T, Cell Signaling Technology, Danvers, MA), goat antibody against mouse IL-1b (AF-401-NA, R&D systems, Minneapolis, MN). HRP conjugated secondary antibodies were used. Western Lightning Plus-ECL chemiluminescence substrate (PerkinElmer, Waltham, MA) was used and blots were developed on film or using a ChemiDoc MP System (Bio-Rad, Hercules, CA). Blots were stripped using 0.2 M NaOH then washed in ddH2O and TBST before blocking and reprobing for actin as a loading control, using an HRP conjugated rabbit antibody against β-actin (13E5, Cell Signaling Technology, Danvers, MA).
Western lightning plus ecl chemiluminescence substrate
Manufactured by PerkinElmer
Sourced in United States
Western Lightning Plus-ECL is a chemiluminescence substrate used for the detection of proteins in western blotting applications. It is designed to provide a sensitive and reliable signal for the visualization of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc mp system, by Bio-Rad (5 mentions)
Criterion tris hcl gel, by Bio-Rad (3 mentions)
Af 401 na, by R&D Systems (1 mentions)
D2u3t, by Cell Signaling Technology (1 mentions)
Nb100 479, by Novus Biologicals (1 mentions)
Protease phosphatase inhibitor cocktail, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by PerkinElmer (1 mentions)
Protease and phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Nitrocellulose membrane, by Abcam (1 mentions)
Ponceau s solution, by Merck Group (1 mentions)
Whatman, by Cytiva (1 mentions)
β actin a1978, by Merck Group (1 mentions)
Socs3, by Cell Signaling Technology (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Mini protean tgx precast polyacrylamide gel, by Bio-Rad (1 mentions)
Image studio lite software, by LI COR (1 mentions)
Vimentin, by Santa Cruz Biotechnology (1 mentions)
Sc 7269, by Santa Cruz Biotechnology (1 mentions)
Bca assay kit, by Bio-Rad (1 mentions)
9 protocols using western lightning plus ecl chemiluminescence substrate
1
Western Blot Analysis of HIF-1α and IL-1β
2
Western Blot Analysis of Infected BMDM
3
Quantifying IL-36γ Expression in Neutrophils
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Ly6G+ neutrophils, isolated by flow cytometry, were homogenized in extraction buffer (50 mM Tris-HCl at pH 7.4, 150 mM NaCl, 1 % Triton X-100, 1 mM ethylenediaminetetraacetic acid, 1 mM ethylene glycol tetraacetic acid, 2 mM Na pyrophosphate, 10 mM Na β-glycerophosphate, 1 mM Na orthovanadate, 1 mM phenylmethanesulfonylfluoride, 1× protease and phosphatase inhibitor cocktail [Sigma]). The protein samples (50 μg) were resolved in a 12 % SDS-PAGE Mini-Protean Precast gel (Bio-Rad) and transferred to a polyvinylidene difluoride membrane (PerkinElmer). The membrane was blocked in PBS containing 0.1 % Tween 20 and 7 % non-fat milk, and then incubated at 4 °C overnight with an antibody against IL-36γ (1:200, Santa Cruz, sc-168163) or β-actin (1:50000, Abcam, mab150), followed by 1 h at room temperature in the appropriate secondary horseradish peroxidase-conjugated antibody (Cell Signaling Technology). The antibodies were detected using the Western Lightning Plus-ECL chemiluminescence substrate (Perkin Elmer).
4
BMDM Protein Extraction and Analysis
5
Western Blot Protein Transfer Optimization
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Proteins were transferred to a nitrocellulose membrane (Abcam) using a semi-dry western blot system. Prewet membrane and gel were sandwiched between Whatman papers that were presoaked in a transfer buffer. A transfer was performed for 45 min at room temperature (RT). A flow rate of electric charge was dependent on length (L), width (W), and amount (n) of membranes, and was calculated as follows: mA=L × W × 2.5 × n. An efficient transfer of proteins was determined by staining a membrane with Ponceau S solution (Sigma-Aldrich, Cat No. 6226-79-5). Afterwards a membrane was incubated in a blocking buffer shaking at RT for 1 h, followed by binding with primary antibody in 5% BSA/TBS-T buffer overnight at 4°C. The following antibodies were used for immunodetection of histone marks: acetylated-H3 (1:20,000), acetylated-H3K9 (1:5,000), and acetylated-H3K9/K14 (1:2,000). Anti-FLAG-tag antibody (1:1,000) was used to detect plant-produced FLAG-HDA6. The membrane was washed three times for 5 min with 1× TBS-T buffer, and incubated for 1 h at RT with a horseradish peroxidase-linked secondary antibody in 5% (w/v) BSA/TBS-T buffer. The membrane was washed once with 1× TBS-T and two times with 1× TBS buffer. The signal was developed using Western lightning plus-ECL chemiluminescence substrate (PerkinElmer, Cat No. NEL105001EA).
6
Immunoblot Analysis of HIF-1α in BMDMs
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BMDMs infected for 12 hours at a multiplicity of infection of 5 as described or peritoneal macrophages infected for 20 hours at a multiplicity of infection of 5 as described were washed with ice cold PBS, lysed in 1X SDS-PAGE buffer on ice and heat sterilized for 20 min at 100°C for removal from the BSL3 facility. Total protein lysate was analyzed by SDS-PAGE using precast 4–20% Criterion TGX protein gels from Bio-Rad Laboratories (5671093), rabbit mAb to HIF-1α (D2U3T) from Cell Signaling Technology (14179S), and an HRP-conjugated secondary antibody. Blots were developed with Western Lightning Plus-ECL chemiluminescence substrate from PerkinElmer (NEL105001EA) and a ChemiDoc MP system from Bio-Rad Laboratories.
7
Mycobacterial Protein Extraction and Western Blot
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Mtb strains were grown to mid-log phase in 7H9 medium. Bacteria were pelleted and washed twice with PBS prior to resuspension in lysis buffer (50 mM Tris-HCl pH 7.5, 5 mM EDTA, 0.6% SDS, 1 mM PMSF). Samples were lysed using a bead-beater, and cell debris were pelleted. Clarified lysates were heat-sterilized at 100°C for 15 min and frozen prior to use. Total protein lysates were analyzed by SDS-PAGE using precast Tris-HCl 4–20% criterion gels (Bio-Rad). Primary polyclonal antibodies for Cfp29 were generated by GenScript USA Inc via immunization of rabbits with three peptides from the protein sequence. HRP-conjugated goat anti-rabbit IgG secondary antibodies were used (sc-2030; Santa Cruz Biotechnology). Western Lightning Plus-ECL chemiluminescence substrate (Perkin Elmer) was used, and blots were developed using a ChemiDoc MP System (Bio-Rad).
8
Immunoblot Analysis of Cellular Proteins
9
Evaluating Inflammatory Protein Markers in Osteoarthritis
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This approach was utilized to determine the protein expression of P2X7R, NLPR3, Pro-Caspase-1, Cleaved caspase-1, p-NF-κB p65, and IκB in cartilage tissues of the knee joint of osteoarthritic-treated and untreated rats. Knee Cartilages were excised, homogenized, and macerated in lysis buffer. Then, supernatant was obtained by centrifugation at 12,000 xg for 5 min at 4 °C. The BCA assay kit (Bio-Rad Labs assay kit, USA) was used to determine the total amount of proteins. Furthermore, 30 µg of protein was incubated for 20 h with the primary antibodies mentioned in Table 1 . This was followed by incubation with goat anti-rabbit-horseradish peroxidase (HRP) secondary antibody. A Chemi-Doc imager was used to acquire the band intensities after applying the Western Lightning Plus ECL Chemiluminescence substrates (Perkin Elmer, USA).
Primary and Secondary antibodies used in the western bolt analysis
| Primary antibodies against | |
| P2X7R | Cat no. # PA5-29,274, Invitrogen, Thermofisher Scientific, USA |
| NLPR3 | Cat no. # ab263899, Abcam, USA |
| Pro-Caspase-1 | Cat no. # 2225, Cell Signaling Technology, USA |
| Cleaved caspase-1 | Cat no. #67,314, Cell Signaling Technology, USA |
| p-NF-κB p65 | Cat no. # ab76302, Abcam, USA |
| IκBα | Cat no. # 4812; Cell Signaling Technology, USA |
| β-actin | Cat no. # sc-47778, Santa Cruz, USA |
| Secondary antibodies against | |
| Horse radish peroxidase (HRP) | Cat no. # 31,460, Thermofisher Scientific, USA |
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