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West pico chemiluminescence substrate

Manufactured by Thermo Fisher Scientific
Sourced in Germany

The West Pico chemiluminescence substrate is a detection reagent used in western blot analysis to visualize protein bands. It produces a luminescent signal in the presence of horseradish peroxidase (HRP) enzyme, which can be detected and quantified using a luminometer or X-ray film.

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13 protocols using west pico chemiluminescence substrate

1

Western Blot Protocol for Cas9 Protein Detection

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Cells were transfected as described above, and protein lysates were prepared 72 hours post transfection. The protein concentrations were determined using an amido black assay. Protein samples were separated onto 4–15% Mini-PROTEAN® TGX Stain-Free™ Protein Gels (Biorad). Proteins were then transferred 1 hr onto a polyvinylidene fluoride membrane. That membrane was blocked for 2 h in 5% milk in 1X Tris-buffered saline (TBS) and 0.01% Tween 20 at room temperature. The CjCas9 and SpCas9 protein fused with HA-tag were detected using anti-HA tag (1:1,000; Cell Signaling Technology, Danvers, MA) and β-actin with anti-β-actin (1:1,000; Cell Signaling Technology) antibodies, respectively, and visualized with HRP conjugated anti-rabbit secondary antibody (1:20,000; Dianova, Hamburg, Germany) using West Pico Chemiluminescence substrate (Thermo Fisher Scientific, Waltham, MA). The revelation was made at different times of exposition to visualise all possible bands on the western.
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2

Quantitative Protein Analysis in Neuronal Cells

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Human neuronal cell pellets or cortical brain tissues were lysed with CellLytic-M (Sigma) for 30 min at 4 °C, and centrifuged at 14000×g. The concentrations of protein from cell lysates or brain tissue homogenates were estimated by bicinchoninic acid (BCA) method (Thermo Scientific, Rockford, IL). Protein load was 20 μg/lane in 4–15% SDS-PAGE gradient gels (Thermo Scientific). Molecular size separated proteins were then transferred onto nitrocellulose membranes, blocked with superblock (Thermo Scientific), and incubated overnight with respective primary antibody to THTR1, THTR2, TPK1, PDHE1α, PDK1, p-PDHE1αSer293, PDH phosphatase and b-actin at 4 °C, followed by washes and incubation with horse-radish peroxidase conjugated secondary antibodies for 1 h at 4 °C.
Immunoreactive bands were detected by West Pico chemi-luminescence substrate (Thermo Scientific). Data were quantified as arbitrary densitometry intensity units using the ImageJ software package. All information about the source of antibodies, intended biomarker, catalog numbers, dilutions factors for immunohistochemical staining, and western blotting analyses are listed in Table 1.
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3

Western Blot Analysis of NLRP3, Caspase-1, and IL-1β

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The hBECs cultured in T-75 cm2 flasks were lysed with CellLytic-M (Sigma) at 4°C. Then cell lysates orbrain tissuehomogenates from animal studies were centrifuged at 14000 × gat 4°C, and then total protein concentrations in respective samples were estimated by BCA (Thermo Scientific, Rockford, IL). We loaded 20 g protein/lane and resolved the various molecular weight proteins by SDS-PAGE on gradient gels (Thermo Scientific) and then transferred the protein onto nitrocellulose membranes. After blocking, membranes were incubated for overnight with primary antibody such as mouse anti-NLRP3 (1:1000), rabbit-anti-caspase-1 (1:2000) and rabbit anti-IL-1 (1:250) at 4°C followed by 1 hr incubation with horse-radish peroxidase conjugated secondary antibodies. Immunoreactive bands were detected by West Pico chemiluminescence substrate (Thermo Scientific). Data were quantified as arbitrary densitometry intensity units using the Gelpro32 software package (Version 3.1, Media Cybernetics, Marlow, UK).
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4

Western Blot Analysis of Mig6 Protein

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Pelleted cells were lysed on ice with RIPA lysis buffer (Thermo Scientific, Rockford, IL) supplemented with protease/phosphatase inhibitors (Roche, Basel, Switzerland). Protein concentrations were determined by the BCA method and lysates diluted in SDS sample buffer (Bio-Rad, Hercules, CA) prior to SDS-PAGE. Anti-Mig6 antibody was a gift from Dr. Ferby (25 (link)). β-actin was obtained from Abcam (Cambridge, MA). All other antibodies were obtained from Cell Signaling (Beverly, MA). Secondary horseradish peroxidase (HRP)-conjugated antibodies were from KPL (Gaithersburg, MD) and signals developed using West-Pico chemiluminescence substrate (Thermo Scientific). ImageJ software was used to quantify immunoblot signals on exposed films.
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5

ZFN Protein Expression Analysis

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Protein detection from cell lysates and concentrated retroviral supernatants was performed as previously described34 (link) with minor adjustments. Briefly, U2OS-EGFP cells transfected with ZFN expression plasmids were harvested 48 h after transfection, while 293T producer cells were collected 60 h post-transfection. Cells were lysed in 100 ml of ice-cold RIPA buffer composed of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% sodium dodecyl sulphate (SDS), 0.5% sodium deoxycholate, 1% Nonidet P-40 and a cocktail of protease inhibitors (Complete Mini, Roche Applied Science). Samples were separated by SDS-polyacrylamide gel electrophoresis (12%) and transferred to polyvinylidene difluoride membranes. ZFN and EGFP expression was detected using antibodies directed against the HA tag (NB600-363; Novus Biologicals, Littleton, CO) and EGFP (MAB3580; Millipore, Billerica, MA), respectively. Control staining was performed using anti-α-actin (sc-81178, Santa Cruz Biotechnology) or anti-p30 antibodies (kindly provided by Sandra K. Ruscetti, NCI Frederick, MA, USA). Proteins were visualised after staining with HRP-conjugated mouse anti-rabbit, (Dianova) mouse anti-rat (Dianova), or donkey anti-goat (Santa Cruz) secondary antibody using the WestPico Chemiluminescence substrate (Thermo Scientific).
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6

Western Blot Analysis of Cortical Proteins

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Cortical brain tissues were lysed with CellLytic-M (Sigma) for 30 min at 4°C, centrifuged at 14,000× g. The protein concentration in homogenate was estimated by bicinchoninic acid (BCA) method (Thermo Scientific, Rockford, IL). Subsequently, 20 μg of protein per lane was loaded into 4–15% SDS-PAGE gradient gels (Thermo Scientific). Proteins separated according to their molecular size were then transferred onto nitrocellulose membranes, blocked with SuperblockT20 (Thermo Scientific), and incubated overnight with respective primary antibody at 4 °C. Incubation with horse-radish peroxidase conjugated secondary antibodies for 1 hour, was followed by detection of immunoreactive bands by West Pico chemiluminescence substrate (Thermo Scientific). For densitometric quantitation of western blots, images were digitized using a BioRad GS800 calibrated densitometer, and analyzed with BioRad Quantity One software.
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7

Protein Quantification and Western Blotting

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Fresh brain tissue was homogenized with CellLytic-M (Sigma) lysis buffer (500 μL buffer for every 10 mg of tissue) on ice using sonication. After 30 min incubation on ice, this homogenization was centrifuged at 13,000 rpm for 20 min at 4 °C and supernatant was collected and assessed for protein concentration by the bicinchoninic acid (BCA) method (Thermo Fisher). Protein was loaded 20 μg/lane in 4–15% SDS-PAGE gradient gels (Thermo Fisher), transferred on the PVDF membranes, blocked with 5% milk, incubated overnight with primary antibodies at 4 °C, washed, and incubated with respective horse-radish peroxidase conjugated secondary antibodies (1:10000 dilution) for 1 h at room temperature. Immunoreactive bands were detected by West Pico chemiluminescence substrate (Thermo Fisher). Data was quantified as densitometry intensity using ImageJ software.
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8

Western Blot Analysis of CXCL12 and CXCR4

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The DRG of CCD mice (n = 4) were combined as one sample for western blot, the DRG of control mice (n = 4) were combined as the same way. Briefly, L4 and L5 lumbar DRG were dissected in control mice and CCD mice and placed temporarily in liquid nitrogen. Then the samples were homogenized in ice-cold lysis buffer by ultrasonic homogenizer (Cole parmer instruments, USA). The crude homogenates were centrifuged at 4 °C for 15 min at 3 000 rpm, and the supernatants were collected. After the protein concentrations were determined, the samples were heated for 5 min at 99 °C, and 30–60 μg protein was loaded onto 12% SDS–polyacrylamide gels, then electrophoretically transferred onto PVDF membranes. The membranes were blocked with 3% non-fat milk for 1 h and incubated overnight at 4 °C with primary antibody. The following primary antibodies were used: rabbit anti-CXCL12 (1:200, Abcam), rabbit anti-CXCR4 (1:200, Abcam), and mouse anti-β-actin (1:1000, CST). The proteins were detected with horseradish peroxidase-conjugated anti-rabbit secondary antibodies (1:1000, CST), visualized using the supersignal west pico chemiluminescence substrate (Thermo. USA), and exposed in Bio-rad chemiDox-XRS imagine system.
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9

Western Blot Analysis of Brain Tissue

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Fresh brain tissues were collected from the impact/surgery sites and homogenized with CelLytic-M (Sigma) lysis buffer (500 μL buffer for every 10 mg of tissue) on ice using sonication. Then the homogenized tissue was centrifuged at 13,000 rpm for 20 min at 4°C and the supernatant was collected and assessed for protein concentration by bicinchoninic acid (BCA) assay (Thermo Fisher). Tissue lysate was formulated as 1 μg/μL, and then loaded as 20 μg/lane in 4–15% SDS-PAGE gradient gels (Thermo Fisher), transferred on PVDF membranes and blocked with 5% milk. The gel was then incubated overnight with primary antibodies at 4°C, washed, and incubated with horse-radish peroxidase conjugated to secondary antibodies (1:10000 dilution) for 1 h at room temperature. West Pico chemiluminescence substrate (Thermo Fisher) was used to detect immunoreactive bands. Data were analyzed by densitometry with ImageJ.
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10

Immunoblotting of ZFNs/TALENs in HEK293T

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HEK293T cells were PEI transfected, as described above, and harvested at indicated time points. Protein lysates were prepared using RIPA buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% deoxycholate). ZFNs/TALENs or β-actin were detected using anti-HA tag (1:2000; Novus Biologicals) or anti-β-actin (1:2000; Cell Signaling) antibodies, respectively. Detection was performed using Horseradish Peroxidase (HRP)-conjugated anti-rabbit antibody (Dianova) and West Pico chemiluminescence substrate (Thermo Scientific).
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