Total protein lysates from the parental, sodA mutant and complemented strains were separated using 12% SDS-PAGE gels, transferred to PVDF (Hybond-P GE Healthcare) membranes and blocked overnight using 10% skim milk in TTBS. The membranes were incubated for 1h at room temperature with anti-sera against OspA, OspB, OspC, DbpA, BBK32, BosR, lactate dehydrogenase (LDH), FlaB (as a loading control) and RpoS at appropriate dilutions [45 (link)–47 (link)]. The blots were developed following incubation with appropriate dilutions of HRP conjugated anti-mouse or anti-rabbit secondary antibodies using ECL Western blotting reagents (GE Healthcare, Buckinghamshire, UK). The same protocol was used for the detection of GAPDH (glyceraldehyde 6-phosphate dehydrogenase) before and after parental and the sodA negative strains were treated with MV.
Ecl western blotting reagent
Manufactured by GE Healthcare
Sourced in United Kingdom, United States, Germany
ECL Western blotting reagents are a set of chemiluminescent detection reagents designed for the analysis of proteins in Western blot procedures. The reagents are used to visualize and quantify specific proteins that have been separated by gel electrophoresis and transferred to a membrane.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc mp system, by Bio-Rad (4 mentions)
Las 3000 luminescent image analyzer, by Fujifilm (3 mentions)
Nxa931, by Cytiva (3 mentions)
Na934, by Cytiva (3 mentions)
Horseradish peroxidase conjugated anti rabbit igg, by GE Healthcare (2 mentions)
Anti calretinin, by Merck Group (2 mentions)
Super rx fuji x ray film, by Fujifilm (2 mentions)
Goat anti rabbit igg hrp, by Cell Signaling Technology (2 mentions)
Pvdf transfer membrane, by PerkinElmer (2 mentions)
Pierce 660 nm protein assay, by Thermo Fisher Scientific (2 mentions)
Mouse anti actin, by MP Biomedicals (2 mentions)
Secondary anti rabbit igg labelled with horseradish peroxidase, by Cell Signaling Technology (2 mentions)
Bradford reagent, by Bio-Rad (2 mentions)
Pvdf membrane, by GE Healthcare (2 mentions)
Lysis buffer, by Cell Signaling Technology (2 mentions)
Complete edta free protease inhibitor cocktail, by Roche (2 mentions)
β actin, by Merck Group (2 mentions)
Amersham imager, by Cytiva (2 mentions)
Protease inhibitor cocktail, by Serva Electrophoresis (2 mentions)
Hybond p, by GE Healthcare (1 mentions)
40 protocols using ecl western blotting reagent
1
Western Blot Analysis of Borrelia Proteins
2
ADAM23 Immunoprecipitation and Detection
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The homogenate supernatants of A549 cells and their transfectants were immunoprecipitated with rabbit anti‐human ADAM23 antibody (Abcam, Cambridge, UK) or nonimmune rabbit IgG (Dako Cytomation),30 and then subjected to immunoblotting with rabbit anti‐human ADAM23 antibody (Abcam) and horseradish peroxidase‐conjugated secondary antibody (DAKO A/S, Glostrup, Denmark), followed by reaction with ECL western blotting reagents (GE Healthcare Bio‐Sciences, Uppsala, Sweden).
3
Protein Extraction and Immunoblot Analysis
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To isolate proteins, samples were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% SDS (Sigma Aldrich, L3771), 1% NP-40 (Sigma Aldrich, NP-40S), 0.25% sodium deoxycholate (Sigma Aldrich, D6750), 1 mM EDTA, 1 mM sodium fluoride, 1 mM Na3VO4) with a protease inhibitor cocktail (Roche Life Science, 11 697 498 001). For immunoblot analysis, proteins were electrophoresed on SDS-polyacrylamide gels and transferred onto PVDF membranes. The membranes were blocked with 4% skim milk (BD Difco, 232100) in Tris-buffered saline (TBS; 50 mM Tris-Cl, 150 mM NaCl, pH 7.5) containing 0.5% Tween-20 (Sigma Aldrich, P1379; TBST) and subsequently incubated with anti-PRDX1 (Abcam, ab41906), anti-PRDX2 (AbFrontier, LF-PA0007), anti-PRDX3 (AbFrontier, LF-PA0030), anti-PRDX4 (AbFrontier, LF-PA0009), anti-NR1H3/LXRα (Abcam, ab41902), anti-LC3B (Cell Signaling Technology, 2775), anti-SQSTM1/p62 (Abcam, ab56416), anti-CD36 (Santa Cruz Biotechnology, sc-9154), anti-LMNB/laminB (Santa Cruz Biotechnology, sc-6217) and anti-GAPDH (Santa Cruz Biothechnology, sc-25778) primary antibody and horseradish peroxidase-conjugated secondary antibody (Millipore, AP106P, AP124P and AP124P). Immunoreactive bands were detected with ECL™ western blotting reagents (GE Healthcare Life Sciences, RPN2106).
4
Western Blot Quantification of Polyamine Modulon Proteins
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Western blot analysis was performed by the method described in [47 (link)] using horseradish peroxidase-conjugated anti-rabbit IgG (GE Healthcare Bio-Sciences) as a secondary antibody and ECL Western blotting reagents (GE Healthcare Bio-Sciences). Antibodies against the proteins encoded by polyamine modulon were prepared as described previously [21 (link),24 (link),26 (link),27 (link),48 (link)]. The level of protein on the blot was quantified with a LAS-3000 luminescent image analyzer (Fuji Film).
5
Immunoblotting Protein Detection Protocol
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Briefly, whole-cell lysates or immunoprecipitated samples were resolved by 4–20% precast BioRad gels (BioRad). PAGE separated proteins were transferred onto a PVDF membrane at 15V overnight at 4C in a Tris-Glycine Transfer Buffer (25 mM Tris, 192 mM glycine, 10% Methanol) by wet transfer procedure in Mini Trans-Blot cell as per the manufacturer protocol. Protein-bound PVDF membranes were incubated for 2 hr at RT with 5% skimmed milk in TBST buffer (20 mM Tris, 150 mM NaCl, 0.1% tween20). Blocked membranes were incubated overnight with primary antibodies. Later, three washes of 5 minutes each in TBST were performed followed by 2 hr incubation at RT with the 1:5000 or 1:10000 diluted horseradish peroxidase conjugated secondary antibodies. Protein bands were visualized with ECL western blotting reagents (GE Health care, Cat no # RPN2106).
6
Western Blot Analysis of Cellular Proteins
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Whole cellular protein extracts were prepared in 95°C Laemmli sample buffer and mechanically sheared by pressing few times through syringes (26 G). Protein concentration was determined using a Pierce™ 660 nm Protein Assay (Thermo Scientific). A total of 5 µg protein per extract was separated on denaturing 10–20% gradient SDS-PAGE gels. Proteins were transferred on PVDF transfer membranes (0.45 µm, Perkin Elmer). Membranes were probed with the following primary antibodies: anti-calretinin (Sigma, HPA007306), mouse anti-actin (#69100) from MP Biomedicals, N-Cadherin (BD Biosciences, 610920), YAP (Cell Signaling 4912), Mesothelin (Rockland Inc. 200-301-A88), GATA4 (C-20) (Santa Cruz sc-1237), p62 (Progen GP62-C), LC3B (Cell Signaling 2775S), p53 (DO-1) (Santa Cruz sc-126), Thy1 (H-110) (Santa Cruz sc-9163), and γ-H2AX (Millipore 05-636). Membranes were then incubated with the secondary antibody rabbit anti-mouse IgG-HRP (A-5420) from Antell, and goat anti-rabbit IgG-HRP (#7074) from Cell Signaling. The signals were detected by enhanced chemiluminescence (ECL™ Western Blotting Reagents, GE Healthcare) and detected on photosensitive film (Super RX Fuji x-Ray Film, Fujifilm). Proteins were quantitated with densitometry using Image J software (Version 1.42q, USA).
7
Western Blot Analysis of NIH 3T3 Cells
8
Ubiquitin Protein Extraction and Detection
9
Western Blot Protocol with RIPA Lysis and ECL Detection
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Cells were lysed with lysis buffer containing radioimmunoprecipitation (RIPA) buffer (50 mM Tris-HCl, pH 8, 2 mM EDTA, 300 mM NaCl, 2% NP-40, 1% sodium deoxycholate) and 1× protease inhibitor (Pierce, Waltham, Massachusetts, USA) and mixed with 1× Laemmli sample buffer (Bio-Rad, Hercules, California, USA) and 5% β-mercaptoethanol (Sigma-Aldrich). The lysates were run on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels. Western blotting was performed using a TransBlot Turbo Transfer System (Bio-Rad) to transfer the protein bands to the polyvinylidene difluoride (PVDF) membrane. Membranes were blocked with 5% milk in phosphate buffered saline, 0.05% Tween 20 (PBST) or tris buffered saline, 0.1% Tween 20 (TBST) for 1 h at room temperature and probed with primary antibodies (S1F Table ) overnight at 4°C. The secondary antibodies (S1F Table ) were added for 1 h at room temperature. Membranes were developed using the ECL Western Blotting Reagents (GE, Amersham, UK) and imaged using the ChemiDoc MP imaging system (Bio-Rad).
10
Identification of Immunogenic Proteins
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The proteins were completely transferred to membranes using the TE70/77 PWR Semi-Dry Transfer Unit (GE Healthcare) according to the manufacturer’s instructions. The membranes were blocked for 1 h at room temperature using 5% rabbit serum in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) and then washed three times for 20 min in TBS-T. The blots were incubated overnight at 4 °C with a 1:500 dilution of the antisera derived from the immune-challenged and control cattle. The blots were then incubated for 1 h at room temperature with a 1:5000 dilution of goat anti-bovine IgG HRP-conjugated antibody (Sigma, USA). After washing, the immunolabeling was detected using ECL Western blotting reagents (GE Healthcare). Finally, specific immunogenic proteins were visualized using a ChemiDoc XRS Camera and the Quantity One 1D analysis software (Bio-Rad).
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