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.
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.
Automatically generated - may contain errors
Lab products found in correlation
Cell lytic m, by Merck Group (4 mentions)
Gradient sds page gel, by Thermo Fisher Scientific (4 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Bca method, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Goat anti rabbit hrp conjugate, by GE Healthcare (2 mentions)
Pierce bca assay, by Thermo Fisher Scientific (2 mentions)
Mini protean tgx stain free protein gel, by Bio-Rad (1 mentions)
Anti ha tag, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Superblock, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid (bca), by Thermo Fisher Scientific (1 mentions)
Sds sample buffer, by Bio-Rad (1 mentions)
β actin, by Abcam (1 mentions)
Protease phosphatase inhibitor, by Roche (1 mentions)
Mab3580, by Merck Group (1 mentions)
Anti α actin, by Santa Cruz Biotechnology (1 mentions)
Nb600 363, by Novus Biologicals (1 mentions)
Complete mini, by Roche (1 mentions)
Gs 800 calibrated densitometer, by Bio-Rad (1 mentions)
13 protocols using west pico chemiluminescence substrate
1
Western Blot Protocol for Cas9 Protein Detection
2
Quantitative Protein Analysis in Neuronal Cells
3
Western Blot Analysis of NLRP3, Caspase-1, and IL-1β
4
Western Blot Analysis of Mig6 Protein
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).
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.
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.
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.
9
Western Blot Analysis of Brain Tissue
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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