For the WB experiments, ciGEnCs were stimulated for 30 min, proteins were extracted using RIPA buffer and protein concentrations were determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). 15 μg of protein was diluted in NuPAGE LDS Sample buffer (4x, Invitrogen) at a final 1x concentration with 50 μM dithiothreitol (DTT, Sigma-Aldrich). The protein samples were loaded into 50 μl 12% 10-well precast polyacrylamide gels and transferred to PVDF membranes (Bio-Rad) using the Trans-Blot Turbo Transfer System (Bio-Rad). Antibodies were used for the detection of human beta-actin (β-actin) and Iκ-Bα. Following gel transfer, membranes were blocked in TBS-T with 5% milk for 1 h, were incubated with the primary antibodies overnight at 4 °C, washed in TBS-T and incubated with secondary antibodies for 1 h (room temperature). Protein detection was achieved using enhanced chemiluminescence (ECL) reagents (Li-COR). Li-COR software was used for protein band densitometry. Due to β-actin and Iκ-Bα having a similar molecular weight, the samples were loaded and were run twice onto two different gels, which were blocked for either β-actin or Iκ-Bα (full-length images are presented on Supplementary Fig. 3 ).
Ecl reagent
Manufactured by LI COR
Sourced in United States
The ECL reagent is a chemiluminescent substrate used for the detection of proteins in Western blot analysis. It produces a luminescent signal when combined with horseradish peroxidase (HRP), which is commonly used as a reporter enzyme in immunoassays. The intensity of the luminescent signal is proportional to the amount of target protein present in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Trans blot turbo apparatus, by Bio-Rad (2 mentions)
Chemidoc apparatus, by Bio-Rad (2 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Nupage novex 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Blocking buffer, by LI COR (1 mentions)
Nupage transfer system, by Thermo Fisher Scientific (1 mentions)
Nupage loading buffer, by Thermo Fisher Scientific (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Odyssey ir scanner, by LI COR (1 mentions)
Ab76026, by Abcam (1 mentions)
Ab137550, by Cell Signaling Technology (1 mentions)
C digit blot scanner, by LI COR (1 mentions)
Sc 133134, by Santa Cruz Biotechnology (1 mentions)
Bradford assay, by Merck Group (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Phospho erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
7 protocols using ecl reagent
1
Western Blot Analysis of Endothelial Cells
2
Cell Lysis and Protein Analysis Protocol
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Lysates for analysis from various cells were prepared with 1X Cell Lysis Buffer (Cell Signaling Technology Cat #9803) containing 20 mM Tris-HCl (pH 7.5),150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4 and1 μg/ml leupeptin with addition of 1 mM PMSF. The lysed cells were collected and centrifuged at 4°C at 13K for 10 mins. The lysate was transferred to a new tube and mixed with NuPAGE loading buffer (NP0007—Invitrogen). Lysates were heated for 10 mins at 70°C and subjected to gel electrophoresis on a NuPAGE Novex 4–12% Bis-Tris Gel (Invitrogen) by following manufacturer’s instructions and transferred to a nitrocellulose membrane using NuPAGE transfer system (Invitrogen). After transfer, the membrane was blocked with blocking buffer (Licor Bioscience), washed and blotted with indicated antibodies at 4°C for 24 hours. Next, the blot was incubated with appropriate secondary HRP/IR-dye-conjugated antibodies were incubated for 45 minutes. After further washing with TBST, the membrane was developed using ECL reagents or IR reader (Licor Bioscience).
3
Western Blot Transfer and Antibody Incubation
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Western blots were performed in modified Towbin buffer consisting of 25 mM Tris, 192 mM glycine, 4 mg/L SDS, 10% (v/v) methanol, and proteins were transferred onto PVDF membrane at 35 V for 2 hr, on ice. Membranes were blocked in 5% (w/v) non-fat milk powder in phosphate-buffered saline (PBS) for 1 hr at 25°C before incubating overnight in diluted primary antibody in 5% (w/v) non-fat milk powder in PBST (PBS containing 0.05% (v/v) Tween-20) at 4°C. Overnight incubated membranes were washed in PBST before incubating with IR-dye conjugated secondary antibody or HRP-conjugated secondary antibody in 5% (w/v) non-fat milk powder containing PBST for 1 hr at 25°C. After incubation with secondary antibodies, the membranes were washed first in PBST and then PBS before scanning on a Li-COR Biosciences (Lincoln, NE) Odyssey IR scanner or developing with ECL reagents.
4
Western Blot Analysis of B. burgdorferi
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Cultures were pelleted at 4°C, 3,200 x g for 20 minutes. B. burgdorferi were washed twice with HN buffer and subsequently lysed in lysis buffer (4% SDS, 0.1M Tris-HCl). SDS-PAGE was performed on the Mini-Tetra System (Bio-Rad, Hercules, CA, United States) using AnykD or 12% polyacrylamide gels and transferred to PVDF membranes using the Transblot Turbo apparatus (Bio-Rad, Hercules, CA, United States). PVDF membranes were blocked for 1-hour in TBST with 5% milk. Commercially available antibodies for OspC (Rockland Immunochemicals, Pottstown, PA), or rabbit polyclonal anti-DksA [11 (link)], anti-BBD18 [56 (link)], anti-RpoS antibody were incubated with the PVDF membranes overnight at a dilution of 1:2000, 1:2000, 1:500, or 1:500, respectively, in TBST. Antibodies bound to the membrane were detected with the incubation of anti-rabbit HRP-conjugated secondary antibodies for 1 hour followed by five washes in TBST. Images were produced by the ChemiDoc apparatus (Bio-Rad, Hercules, CA, United States) using ECL reagent (LI-COR, Lincoln, NE, United States). Uncropped and replicate images of westernblots are available in S2 Text .
5
Cytokine-Induced Protein Profiling
6
Western Blot Analysis of HUVEC Proteins
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For western blot analysis, HUVECs were lysed in RIPA buffer (Sigma-Aldrich) with 10% protease and 1% phosphatase inhibitors (Sigma-Aldrich). Protein content was determined using the Bradford assay (Sigma-Aldrich), and 50 µg of lysates was separated by electrophoresis using 4–12% PAGE gels (Lonza) and transferred onto a PVDF membrane (Perkin Elmer, Waltham, MA, USA). After membranes were blocked with 5% non-fat dried milk or 5% bovine serum albumin, they were incubated with primary antibodies overnight at 4 °C: phospho-NRF2-Ser40 (1:1000) and NRF2 (1:1000) (Abcam, ab76026 and ab137550), phospho-ERK1/2-Thr202/Tyr204 (1:1000) and ERK1/2 (1:1000) (9101 and 9102, Cell Signaling), and SOD2 (1:500; sc-133134, Santa Cruz Biotechnology, CA, USA). Blots were revealed by LI-COR ECL Reagent and by a C-DiGit Blot scanner (LI-COR Biosciences) after incubation with appropriate secondary horseradish peroxidase-conjugated IgG antibodies (GE Healthcare Europe GmbH, Milan, Italy) at a 1:3000 dilution for 1 h at room temperature. Proteins were quantified using Image Studio software (http://www.licor.com ). Human ACTIN-b (1:1000) (Sigma-Aldrich) was used as a loading control.
7
Western Blot Analysis of B. burgdorferi
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Cultures were pelleted at 4 °C, 3,200 x g for 20 minutes. B. burgdorferi were washed twice with HN buffer and subsequently lysed in lysis buffer (4% SDS, 0.1M Tris-HCl). SDS-PAGE was performed on the Mini-Tetra System (Bio-Rad, Hercules, CA, United States) using AnykD or 12% polyacrylamide gels and transferred to PVDF 105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC
The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10.1101/2020.11.04.367946 doi: bioRxiv preprint membranes using the Transblot Turbo apparatus (Bio-Rad, Hercules, CA, United States). PVDF membranes were blocked for 1-hour in TBST with 5% milk.
Commercially available antibodies for OspC (Rockland Immunochemicals, Pottstown, PA), or rabbit polyclonal anti-DksA (11) , anti-BBD18 (56), anti-RpoS antibody were incubated with the PVDF membranes overnight at a dilution of 1:2000, 1:2000, 1:500, or 1:500, respectively, in TBST. Antibodies bound to the membrane were detected with the incubation of anti-rabbit HRP-conjugated secondary antibodies for 1 hour followed by five washes in TBST. Images were produced by the ChemiDoc apparatus (Bio-Rad, Hercules, CA, United States) using ECL reagent (LI-COR, Lincoln, NE, United States).
(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC
The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10.1101/2020.11.04.367946 doi: bioRxiv preprint membranes using the Transblot Turbo apparatus (Bio-Rad, Hercules, CA, United States). PVDF membranes were blocked for 1-hour in TBST with 5% milk.
Commercially available antibodies for OspC (Rockland Immunochemicals, Pottstown, PA), or rabbit polyclonal anti-DksA (11) , anti-BBD18 (56), anti-RpoS antibody were incubated with the PVDF membranes overnight at a dilution of 1:2000, 1:2000, 1:500, or 1:500, respectively, in TBST. Antibodies bound to the membrane were detected with the incubation of anti-rabbit HRP-conjugated secondary antibodies for 1 hour followed by five washes in TBST. Images were produced by the ChemiDoc apparatus (Bio-Rad, Hercules, CA, United States) using ECL reagent (LI-COR, Lincoln, NE, United States).
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