The quantification of PIA assay was conducted according to the previously described method (Rao et al., 2022 (link)). Briefly, bacteria were diluted 1:100 in 0.5% TSBG with or without ciprofloxacin and incubated in 6-well plates at 37°C for 24 h. Wells were then washed 3 times with PBS to remove planktonic bacteria. Biofilms of S. aureus with or without ciprofloxacin treatment were scraped and resuspended in 500 μL EDTA (0.5 M) (pH 8.0), boiled for 10 min, then centrifuged. Forty microliter of supernatant from each sample was digested with proteinase K (20 mg/mL) at 37°C for 3 h to reduce nonspecific background. PIA extracts were dotted onto methanol-activated PVDF membranes. After air drying, membranes were blocked with 5% skim milk at room temperature for 2 h, washed three times with PBST (0.1% Tween), then incubated with WGA-HRP at 37°C for 1 h. After three more PBST (0.1% Tween) washes, blots were detected and visualized using ECL Western blot substrate (Thermo Scientific, Rockford, IL, United States).
Ecl western blot substrate
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ECL western blot substrate is a chemiluminescent detection reagent used for western blot analysis. It generates a luminescent signal when exposed to the enzyme-labeled target proteins, allowing for the visualization and quantification of specific proteins in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
X ray film, by AGFA HealthCare (2 mentions)
Polyvinylidene fluoride membrane, by Roche (2 mentions)
Rabbit anti p105 p50, by Cell Signaling Technology (2 mentions)
Hrp conjugated secondary antibody, by Merck Group (2 mentions)
Bca protein concentration assay, by Thermo Fisher Scientific (2 mentions)
X ray film, by Fujifilm (2 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions)
Trans blot semi dry transfer cell, by Bio-Rad (1 mentions)
Protease and phosphatase inhibitor cocktail, by Cell Signaling Technology (1 mentions)
P erk5, by Cell Signaling Technology (1 mentions)
Bca method, by Thermo Fisher Scientific (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Ab6789, by Abcam (1 mentions)
Halt protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
β actin, by Abcam (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
30 protocols using ecl western blot substrate
1
Quantification of Staphylococcus aureus Biofilm Polysaccharide Intercellular Adhesin
2
Immunoblotting of Thermophilic Archaeal Proteins
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Cell sample of 15 ml culture was pelleted by centrifugation and re-suspended in 150 μl TBST buffer (50 mM Tris–HCL, 100mM NaCl, 0.1% Tween-20, pH7.6). Sonication of the cell suspension gave total cell extracts, which were fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein bands on the gel were transferred onto a nitrocellulose membrane using a Trans-Blot Semi-Dry Transfer Cell (Bio-Rad). For immunoblotting, the membrane was immerged in 5% skim milk blocking agent, then incubated with individual primary antibodies and finally with corresponding second antibodies. TFB3 antisera were raised against S. solfataricus TFB3 (18 (link)) (kindly provided by Prof. Malcolm F. White) whereas PCNA3 antisera against S. islandicus PCNA3 were reported previously. Secondary antibodies were purchased from Thermo Fisher Scientific, and hybridization signals were detected using the ECL western blot substrate (Thermo Fisher Scientific), and visualized by exposure of the membrane to an X-ray film (Agfa HealthCare, Belgium).
3
Western Blot Analysis of HAECs
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After the indicated stimulation, cell lysates were prepared from HAECs using a commercial cell lysis containing protease and phosphatase inhibitor cocktail (#5872, Cell Signaling Technology, USA). Protein concentration was measured using the BCA method (Thermo Fisher Scientific, USA). Protein from each group (20 μg) was subjected to 10-12% sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were then transferred to a PVDF membrane (Bio-Rad, USA), then blocked with 5 % fat skim milk in TBST. The PVDF membrane was then sequentially probed with primary antibodies overnight at 4° C and horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at RT. Blots were developed with enhanced chemiluminescence (ECL) western blot substrate (Thermo Fisher Scientific, USA) and exposed on X-ray film (#4741019291, Fujifilm). The following antibodies were used in this study: GPR30 (1:1000, #ab39742, Abcam, USA), KLF2 (1:1000, #ab139699, Abcam, USA), p-ERK5 (1:1000, #3371, Cell Signaling Technology, USA), β-actin (#1:10000, #4970, Cell Signaling Technology, USA), anti-rabbit IgG, HRP-linked secondary antibody (1:3000, #7074, Cell Signaling Technology, USA); anti-mouse IgG, HRP-linked antibody (1:3000, #7076, Cell Signaling Technology, USA).
4
Western Blotting of Protein Markers
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Cell lysates were prepared using RIPA lysis buffer supplemented with protease and phosphatase inhibitors (Complete Protease Inhibitor Cocktail, Roche, Basel, Switzerland and Halt Protease and Phosphatase Inhibitor Thermo Scientific, Rockford, IL, USA) as previously described [29 (link)]. Primary antibodies used included AR-V7 (31-1109-00, RevMab Biosciences, San Francisco, CA, USA), γH2A.X (05-636, Merck Millipore, Darmstadt, Germany), and β-Actin (ab6276, Abcam, Cambridge, UK). As a secondary antibody, a horseradish peroxidase-conjugated antibody (ab6789, Abcam) was used. Signals were detected using ECL Western Blot Substrate or SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, Rockford, IL, USA) on a Fusion S imaging system (Vilber Lourmat, Radolfzell, Germany).
5
Western Blot Analysis of Neu and GAPDH
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Cells and tissues were lysed in triton lysis buffer (25 mM sodium phosphate, 150 mM sodium chloride, 1% Triton X-100, 5 mM EDTA, 50 mM sodium fluoride, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 5 μM pepstatin A, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 25 μM phenylarsine oxide) for 30 min at 4 °C. Aliquots of 50 micrograms in Laemmli buffer were heated in boiling water for 5 min, electrophoresed on 10% polyacrylamide gels, and transferred to PVDF membranes (Biorad, Hercules, CA). The membranes were treated for one hour at room temperature with blocking buffer (5% milk proteins, 0.05% Tween 20 in Tris buffer, pH 8.1) and hybridized overnight in the same buffer containing 1:500 dilutions of rabbit anti-Neu (SC-284) and rabbit anti-GAPDH (SC-25,778) (both antibodies from Santa Cruz Biotechnology, Santa Cruz, CA). The membranes were washed 3 times for 5 min with 0.05% Tween-20 in Tris-Cl, pH 8.1 and probed with a 1:2000 dilution of goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology, SC-2004) in blocking buffer for 1 h at room temperature. The membranes were then washed 3 times for 10 min in 0.05% Tween 20 in Tris buffer, pH 8.1 and incubated with ECL Western blot Substrate (ThermoFisher, Grand Island, NY, Catalog Number 32106) for 1 min before being exposed to X-ray films (Denville Scientific, Holliston, MA, Catalog Number E3012) for 5–10 min and developed.
6
Western Blot Analysis of Autophagy Markers
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Cells were lysed using cell lysis buffer. The proteins were subjected to polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (Roche Diagnostics, Roswell, GA). LC3 (1:1000, 4 °C, 12 h) and E2 (1:500, 4 °C, 12 h) polyclonal antibodies and Beclin1 (1:500, 4 °C, 12 h) and GAPDH (1:1000, 4 °C, 12 h) monoclonal antibodies were used as primary antibodies in combination with the appropriate peroxidase-labelled secondary antibodies. Proteins were detected using an enhanced chemiluminescent ECL western blot substrate (Thermo, Waltham, MA). The images were acquired using the ChemiDoc XRS+ station western blotting detection system (Bio-Rad Laboratories, Inc., Berkeley, CA). Quantification of the protein blots was performed using ImageJ2x software.
7
Western Blot Analysis of ASNS, RARS, and β-Actin
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Protein lysates were extracted with ice-cold RIPA buffer supplemented with protease and phosphatase inhibitors (Roche). Thirty μg of protein lysates were separated using SDS–polyacrylamide gel electrophoresis and transferred to a PVDF membrane (Immobilion-P, Millipore, IPVH00010). After blocking the membranes in 5% BSA in TBST (1× TBS (Cell Signalling); 0.1% Tween20 (Sigma)), the membranes were incubated overnight at 4 °C with primary antibodies either rabbit anti-ASNS antibody (Proteintech, 14681–1-AP) diluted 1:1,000 in 5% BSA (Sigma), mouse anti-β-Actin antibody (Millipore Sigma A5441) diluted 1:5,000 in 5% BSA (Sigma), and rabbit anti-RARS (Proteintech, 27344–1-AP) diluted 1:1,000 in 5% BSA (Sigma).
Primary antibodies were incubated in 5% BSA in TBST overnight at 4 °C. After washing the blots 3 times for 15 min each in TBST, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG (H+L) or HRP-conjugated goat anti-mouse IgG (H+L) secondary antibody (Invitrogen). Finally, the membranes were incubated with ECL western blot substrate (Thermo Scientific) for 1 min. X-Ray films (Fujifilm) were exposed to the western blot membranes and developed with a film processor (SRX-101A, Konica Minolta) and exposure.
Primary antibodies were incubated in 5% BSA in TBST overnight at 4 °C. After washing the blots 3 times for 15 min each in TBST, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG (H+L) or HRP-conjugated goat anti-mouse IgG (H+L) secondary antibody (Invitrogen). Finally, the membranes were incubated with ECL western blot substrate (Thermo Scientific) for 1 min. X-Ray films (Fujifilm) were exposed to the western blot membranes and developed with a film processor (SRX-101A, Konica Minolta) and exposure.
8
Protein Extraction and Western Blot Analysis
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Cells washed with PBS and mouse brains after dissection were solubilized with lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4) supplemented with 1 % Triton X-100 (Sigma-Aldrich) and protease inhibitor cocktail (Sigma-Aldrich) and then cleared at 14,000 g at 4 °C for 30 min. Protein concentrations were determined using the BCA protein concentration assay as manufacturer’s instructions (Thermo Scientific). Subsequently, proteins were separated by electrophoresis onto 4–20 % SDS-PAGE gels and then transferred onto Immobilon-P membrane. Membranes were incubated 1 h at room temperature (RT) with the following antibodies: rabbit anti-LRRK2 MJFF2 (1:1000, Abcam), rabbit anti-IL1β (1:1000, Santa Cruz), rabbit anti-COX-2 (1:2000, Cayman Chemical), mouse anti-GAPDH (1:2000, Millipore), mouse anti-β-tubulin (1:3000, Sigma-Aldrich), mouse α-actin (1:3000, Sigma-Aldrich), rabbit anti-p65 total (1:2000, Cell signaling), rabbit anti-phospho serine 536 p65 (1:1000, Cell signaling), rabbit anti-p105/p50 (1:2000, Cell signaling), and rabbit anti-phospho serine 337 p50 (1:1000, Santa Cruz). Subsequently, membranes were incubated 1 h at RT with HRP-conjugated secondary antibodies (Sigma-Aldrich) and finally incubated with ECL western blot substrate (Thermo Scientific).
9
Molecular Changes in MCAO/R Mouse Model
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At 48 hours post-MCAO/R, mice were deeply anesthetized and the ipsilesional cortex and hippocampal regions were collected (Bregma: -1.5 mm to 3.5 mm). Total protein was isolated by homogenization in cold NP-40 lysis buffer (Boston Bioproducts Inc., Boston, MA, USA). Supernatants were collected after centrifugation at 12,000 rpm for 15 minutes. A total of 30 μg protein was loaded onto 4% to 12% Bis-Tris NuPAGE Novex gels (Invitrogen) and transferred onto NC membranes (Invitrogen). Primary antibodies used include the following: MMP-9 (1:1000, Abcam), Iba-1 (1:500, Abcam), CD11b (1:1000, Abcam), ZO-1 (1:500, Invitrogen), BDNF (1:1000, Abcam), and β-actin (1:2500, Thermo Fisher Scientific, Carlsbad, CA, USA). (Invitrogen, Carlsbad, CA, USA) Blots were probed with appropriate horseradish peroxidase-conjugated secondary antibodies (1:3000, Invitrogen) and detected using the ECL Western blot substrate (Thermo Fisher Scientific). Protein band intensities were quantified using ImageJ.
10
Lobaplatin Alters Cell Cycle and Apoptosis Signaling
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Western blot was used to evaluate the expression level of the proteins of cell cycle and apoptosis-related signaling pathways, including cyclin A2, PARP, Bax, Bcl-2, caspase-3, caspase-8, and caspase-9. SK-MES-1 cells were exposed to various concentrations of lobaplatin for different periods of time, and then were washed twice with cold PBS and lysed in radioimmunoprecipitation assay buffer. The protein concentrations were measured using the bicinchoninic acid method. Total cell proteins were subjected to electrophoresis on 10%–12% polyacrylamide gels, transferred to nitrocellulose membranes, and probed with the following antibodies: cyclin A2, PARP, Bax, Bcl-2, caspase-3, caspase-8, and caspase-9 (1:1,000). The immunoblots were developed and visualized by ECL Western blot substrate (Thermo Fisher Scientific). Glyceraldehyde 3-phosphate dehydrogenase was used as internal control. The test of each group was repeated three times. The image J analysis system was used to measure the intensity of the bands.
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