A549 cells were grown in a 100-mm dish to 90% confluency. After 18 hr, A549 cells were used for co-IP assay of endogenous proteins. For A549 expressing GFP-only, A549 cells were transfected with 12 µg of pEGFP-C2 mixed with 36 µg PEI in DMEM and harvested after 24 hr hours. Cells were harvested with 800 µl NP-40 buffer (20 mM Tris pH = 8, 150–200 mM NaCl, 0.5 mM EDTA, 1% NP-40, 1 mM DTT, 5% glycerol and protease inhibitor cocktail; Sigma-Aldrich). protease inhibitor cocktail (Bimake, #B15002) was added to buffer in experiments precipitating with anti E-cadherin. Cell extracts were sonicated and centrifuged at 4°C for 15 min at 16,000 ×g. The appropriate antibodies were incubated with cell lysate on a rotator at 4°C for 2 h. The lysate-antibodies mix was added to A/G beads prewashed with 300 μl of NP-40 buffer and incubated on a rotator at 4°C for 2 h. Then, the mix was washed three times with NP-40 buffer and SDS-sample buffer was added. Samples were dissolved on SDS-PAGE and analyzed by Western blot. Proteins from Western blots were detected using the EZ-ECL Chemiluminescence Detection kit (Biological Industries), and the band intensity was analyzed using Bio-Rad Gel dox CR Luminescent Image Analyzer and Fujifilm Image Gauge Ver. 3.46 software (Fujifilm, Tokyo, Japan). The ImageGauge software detects saturation to ensure the linearity of the signal.
Ez ecl chemiluminescence detection kit
Manufactured by Sartorius
Sourced in Israel
The EZ-ECL chemiluminescence detection kit is a lab equipment product designed for the detection and analysis of proteins in Western blot applications. It provides a sensitive and reliable method for visualizing target proteins labeled with HRP-conjugated antibodies through a chemiluminescent reaction.
Automatically generated - may contain errors
Lab products found in correlation
Dc protein assay, by Bio-Rad (3 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Anti parp, by Cell Signaling Technology (2 mentions)
Iblot dry blotting system, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Selleck Chemicals (1 mentions)
Iblot gel transfer stacks pvdf membranes, by Thermo Fisher Scientific (1 mentions)
Anti actin hrp, by Santa Cruz Biotechnology (1 mentions)
Anti cdc25a, by Santa Cruz Biotechnology (1 mentions)
Anti wee1, by Santa Cruz Biotechnology (1 mentions)
Anti chk1, by Santa Cruz Biotechnology (1 mentions)
Anti bcl2, by Agilent Technologies (1 mentions)
Anti hif 1α, by Merck Group (1 mentions)
Anti gls, by Proteintech (1 mentions)
Hybond c extra nitrocellulose membrane, by Cytiva (1 mentions)
Trans blot turbo mini pvdf membrane, by Bio-Rad (1 mentions)
21 protocols using ez ecl chemiluminescence detection kit
1
Co-Immunoprecipitation of Endogenous Proteins in A549 Cells
2
Western Blot Analysis of Cell Signaling Proteins
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Cell homogenates were obtained in RIPA buffer (Pierce, Thermo Scientific), supplemented with 1 × EDTA-free complete protease inhibitor cocktail (Roche). Protein concentration was quantified by a modified Lowry assay (DC protein assay; Bio-Rad). 30 μg of protein were resolved in NuPAGE 4-12% Bis-Tris gels and transferred to iBlot Gel Transfer Stacks PVDF membranes (Life Technologies, Thermo Fisher Scientific). After blocking with Tris-buffered saline with Tween-20 containing 5% non-fat dry milk or 5% BSA for 1 h at room temperature, membranes were probed overnight at 4°C with the following antibodies: anti-PARP [1:2000, Cell Signaling #9542]; anti-CDC25A [1:1000, Santa Cruz #sc-7389]; anti-WEE1 [1:1000, Santa Cruz #sc-5285]; anti-CHK1 [1:2000, Santa Cruz #sc-8408]; anti-BCL2 [1:1000, Dako #M0887]; anti-AKT3 [1:1000, Upstate #05-780]; anti-VEGF [1:1000, Abcam #ab46154]; anti-Mouse IgG-Peroxidase antibody produced in rabbit (1:10.000, Sigma-Aldrich #A9044); anti-Rabbit IgG-Peroxidase antibody produced in goat (1:10.000, Sigma-Aldrich #A0545) and anti-Actin HRP [1:40.000, Santa Cruz #sc-1616]. Membranes were developed with SuperSignal Dura detection kit (Pierce) or EZ-ECL Chemiluminescence detection kit (Biological Industries, Kibbutz Beit-Haemek, Israel).
3
Western Blot Detection of Smc1-AID-6xFLAG
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For the detection of Smc1-AID-6xFLAG by western blot, 50 ml of yeast cells were grown until late-exponential phase. Smc1 depletion was induced by the addition of 1 mM of indole-3-acetic acid (IAA) for 1 h before harvest. Cells were centrifuged and pellets were frozen at −80°C overnight. Next, cell pellets were thawed and incubated in 1 ml of 0.2 M NaOH for 5 min, followed by boiling at 95°C for 5 min in 50 μl of Laemmli Buffer including 10% β-mercaptoethanol. Lysates were centrifuged for 2 min and 10 μl of the supernatants were loaded and run on a 10% SDS-PAGE gel. Separated protein bands were transferred to a PVDF membrane followed by blocking with PBST + 10% skim milk for 1 h. The membrane was washed with PBST once for 5 min, and was incubated with a primary mouse anti-FLAG antibody conjugated with HRP enzyme (1:1000) in PBST + 10% skim milk for 1 h. The membrane was washed three times with PBST and Smc1-AID-6xFLAG was detected with the EZ-ECL Chemiluminescence detection kit (Biological Industries) according to the manufacturer’s protocol. Detection of Pgk1 bands was performed with a primary mouse anti-Pgk1 antibody (1:5000) and a secondary goat-anti-mouse antibody (1:10 000) conjugated with HRP.
4
Western Blot Protocol for Hypoxia Regulators
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Western blot analyses with whole-cell RIPA buffer protein lysates were separated by SDS-PAGE and blotted onto Hybond-C-Extra nitrocellulose membranes (Amersham Bioscience, Little Chalfont, UK) or onto Trans-Blot Turbo Mini PVDF membranes (BioRad). Following primary antibodies were used after blocking: anti-HIF-1α (1:500, rabbit polyclonal, Millipore, CA) anti-HIF-2α (1:500, rabbit polyclonal Abcam), anti-c-Myc (1:100, mouse monoclonal Santa Cruz), anti-GLS (1:500, rabbit polyclonal, Proteintech), anti-SDHA (1:2,000, mouse monoclonal Abcam) and anti-β-Actin (1:500, mouse monoclonal Santa Cruz). The proteins were detected by using HRP-conjugated secondary antibodies; anti-mouse IgG (1:5,000, GE healthcare, Buckinghamshire, UK) and anti-rabbit IgG (1:5,000, GE healthcare) and EZ-ECL chemiluminescence detection kit (Biological Industries, Israel).
5
Quantification of IKK and IκB Proteins
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Total protein was separated by SDS-PAGE and transferred onto PVDF membranes by the iBlot Dry Blotting System (Invitrogen, MA, USA). Blots were incubated overnight at 4 °C with primary anti-human antibodies for phospho-IKK α/β (2697 S, 1:1000; Cell Signaling Technology, MA, USA), IKK α/β (sc-7607,1:400; Santa Cruz Biotechnology, TX, USA) phospho-IKB-α (9246 S, 1:200; Cell Signaling Technology) and IKB-α (sc-203, 1:200; Santa Cruz Biotechnology). Thereafter, the blots were incubated with either donkey anti-rabbit (Biolegend, CA, USA) or anti-mouse (Cell Signaling Technology) HRP-linked antibody (1:2000). To assess housekeeping protein expression, membranes were reblotted overnight at 4 °C with primary rabbit anti-human β-actin HRP conjugate (Cell Signaling Technology). Bands were visualized using the EZ-ECL chemiluminescence detection kit (Biological Industries, Israel). Total IKK α/β, IKB-α and β-actin were used as internal controls when applicable to verify basal level expression and equal protein loading. Further details included as Supplementary Information.
6
Western Blot Assay for MyD88 Protein Detection
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For MyD88 protein detection, 107 cells were lysed in Ripa buffer in the presence of protease inhibitors and incubated on ice for 25 min. The lysates were then centrifuged at 10,000g for 15 min. Proteins were separated by gel electrophoresis and transferred to nitrocellulose membranes. The nitrocellulose membranes were blocked with 5% skim milk for 1 h and MyD88 was detected using 1 µg/mL polyclonal rabbit anti-MyD88 (eBioscience, San Diego, CA, USA) followed by 100 ng/mL goat anti-rabbit IgG-HRP (Abcam, Cambridge, UK). After washing extensively with Tris-buffered saline (TBS) containing 0.2% Tween 20, the membranes were exposed to chemiluminescent substrate in the presence of hydrogen peroxide, using the EZ-ECL-chemiluminescence detection kit (Biological Industries, Israel). A Gel-Doc imaging system (Bio-Rad, Hercules, CA, USA) was used to capture the image.
7
Immunoprecipitation and Western Blot Analysis
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MDA-MB-231 cells were grown in a 100-mm dish to 90% confluency. Cells were harvested with 800 µl NP-40 buffer (20 mM Tris, pH = 8.0, 150–200 mM NaCl, 0.5 mM EDTA, 1% NP-40, 1 mM DTT, 5% Glycerol and Protease inhibitor cocktail; Sigma-Aldrich). Cell extracts were sonicated and centrifuged at 4°C for 15 min at 16,000 × g. The appropriate antibodies were incubated with cell lysate on a rotator at 4°C for 2 h. The lysate-antibodies mix was added to A/G beads or protein L beads (for IP with anti-Scrib) (Santa Cruz Biotechnologies) prewashed with 300 μl NP-40 buffer and incubated on a rotator at 4°C for 2 h. Then, the mix was washed three times with NP-40 Buffer and SDS-sample buffer was added. Samples were dissolved on SDS–PAGE and analyzed by Western blot. Proteins from Western blots were detected using the EZ-ECL Chemiluminescence Detection kit (Biological Industries) and the band intensity was analyzed using Bio-Rad Gel dox CR Luminescent Image Analyzer and Fujifilm Image Gauge Ver. 3.46 software (Fujifilm, Tokyo, Japan). The ImageGauge software detects saturation ensuring the linearity of the signal.
8
Protein Expression Analysis of eIF5A2 and Doxorubicin
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Cells were harvested in cell lysis buffer (Cell Signaling Technology, Inc.) containing protease inhibitors (Sigma-Aldrich; Merck KGaA) following treatment with eIF5A2 siRNA and doxorubicin for 48 h. The protein concentration was determined by BCA assay (Applygen Technologies, Inc.). A total of 10 µg protein was loaded per lane, separated by 15% SDS-PAGE and transferred to 0.45-µm polyvinylidene fluoride membranes (EMD Millipore). The membranes were blocked with TBS-Tween-20 (0.1% Tween-20) containing 0.5% BSA (cat. no. 4240GR100; NeoFroxx GmbH) and incubated with the aforementioned primary antibodies at 4°C overnight. The membranes were washed with TBS-0.1% Tween-20 three times. Following incubation with anti-mouse IgG or anti-rabbit IgG secondary antibodies at 4°C for 2 h, protein expression was detected by chemiluminescence with an EZ-ECL Chemiluminescence Detection Kit (Biological Industries).
9
Neuronal Protein Extraction and Analysis
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Neurons were harvested and washed once with ice-cold PBS, then lysed in triton lysis buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM EDTA, and 1% Triton with a cocktail of protease (Roche) and phosphatase (Sigma-Aldrich) inhibitors for 30 min at 4ºC. Supernatants were obtained after centrifugation at 14 000 × g for 10 min at 4ºC. Protein concentration was quantified by a modified Lowry assay (DC protein assay; Bio-Rad, Hercules, CA, USA). Next, 7–20 μg of protein was resolved by SDS-polyacrylamide gels and transferred onto PVDF (Millipore Ibérica, Madrid, Spain). Membranes were incubated with blocking solution (5% non-fat dry milk in TBS with 0.1% tween-20 (TBS-T)) for 1 h at r.t. Primary antibodies prepared in blocking solution were incubated overnight at 4ºC. After washes with TBS-T, membranes were probed with the appropriate specific peroxidase-conjugated secondary antibody and developed using the EZ-ECL chemiluminescence detection kit (Biological Industries, Kibbutz Beit Haemek, Israel). The primary antibodies used were anti-FAIM-L (antibody generated with the specific sequence presented in FAIM-L) and anti-FAIM-S (both in-house, tested and validated for both WB and immunohisto/cytochemistry23 (link)); anti-caspase-3, anti-PARP (Cell Signalling Technologies, Beverly, MA, USA); anti-NeuN, anti-panERK (Millipore); and anti-tubulin (Sigma-Aldrich).
10
Evaluation of IKK Signaling in pSS
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Protein lysates obtained from equal numbers of PBMC from patients with pSS and healthy donors were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to standard electrophoresis and transfer techniques. Membranes were incubated for 90 min for detection of phospho-IKKε, IKKε, phospho-IKKα/β, phospho-IKKγ, IKKγ, phospho-NF-κB p65, NF-κB p65, IKKα/β, IκBα, and phospho-IκBα using the same antibodies that were used for IHC (see above). Secondary staining was done with secondary antibody-HRP conjugate (MultiSciences (Lianke) Biotech Co., Hangzhou, China), and blots were developed using the EZ-ECL chemiluminescence detection kit (Biological Industries, Kibbutz Beit Haemek, Israel). β-actin was used as protein loading control. Relative IKK protein expression was demonstrated as a ratio (IKK gray scale/β-actin gray scale) by Bio-Rad Quantity One software.
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