Tissues were snap frozen in liquid nitrogen following dissection then thawed and homogenized in lysis buffer using a Polytron homogenizer (T10 basic ULTRA-TURRAX, IKA). Proteins were separated by SDS-PAGE and blotted onto PVDF membranes (Bio-Rad, Munich, Germany). Membranes were incubated with 1% blocking reagent (Roche, Mannheim, Germany) for 1 h, before incubating with primary antibody (1:1,000) diluted in 0.5% blocking solution overnight at 4 °C. After three washing steps with TBS-T the membranes were incubated with the respective secondary antibodies for 1 h at room temperature (peroxidase-coupled anti-rabbit A6154, Sigma–Aldrich, 1:2000, anti-mouse A4416, Sigma–Aldrich, 1:10,000). After three washing steps, the signals were visualized using Pierce ECL Western Blotting Substrate (Perbio Science, Bonn, Germany) and exposure to chemiluminescence film (Amersham, Braunschweig, Germany).
Chemiluminescence film
Manufactured by Cytiva
Sourced in Germany
Chemiluminescence film is a type of photographic film used to detect and record light-emitting chemical reactions, known as chemiluminescence. It is a sensitive film capable of capturing and recording low-level light signals, making it a valuable tool in various applications such as molecular biology, biochemistry, and biomedical research.
Automatically generated - may contain errors
Lab products found in correlation
Anti rabbit a6154, by Merck Group (1 mentions)
Anti mouse a4416, by Merck Group (1 mentions)
Blocking reagent, by Roche (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Pathscan stress and apoptosis signaling antibody array kit, by Cell Signaling Technology (1 mentions)
Sirt5, by Cell Signaling Technology (1 mentions)
Ptm 401, by PTM Biolabs (1 mentions)
Sirt5 antibody, by Cell Signaling Technology (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Bradford protein assay, by Merck Group (1 mentions)
Ab191468, by Abcam (1 mentions)
Anti rabbit igg hrp, by Merck Group (1 mentions)
Hrp substrate, by Cytiva (1 mentions)
Anti mouse igg, by Merck Group (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
5 protocols using chemiluminescence film
1
Western Blot Analysis of Protein Expression
2
Signaling Pathways: Stress and Apoptosis
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For the simultaneous detection of 19 signaling molecules that are involved in the regulation of the stress response and apoptosis, the PathScan® Stress and Apoptosis Signaling Antibody Array Kit has been used according to the manufacturer’s instructions (Cell Signaling Technology, Danvers, MA, USA). Briefly, MCF-7 cells were grown until 80% confluence, treated with the selected compounds, and lysed in 1X Cell Lysis Buffer to collect cell lysates. The array-blocking buffer was added to each well for 15 min at room temperature. Then, 30 μg of solubilized proteins were added to wells and incubated for 2 h at room temperature. Subsequently, the Detection Antibody Cocktail supplied with the kit was added and maintained for 1 h at room temperature. The slide was then incubated for 30 min with horseradish peroxidase-linked streptavidin solution at room temperature. Finally, the slide was covered with LumiGLO/Peroxide reagent (supplied with the kit) and exposed to chemiluminescence film (Amersham Biosciences, Little Chalfont, UK) for 2 to 60 sec. The images were then acquired and the signal intensity was measured using the ImageJ software for Microsoft Windows (National Institutes of Health, Bethesda, MD, USA).
3
Western Blot Analysis of Protein Modifications
4
Verification of Prime Editor Expression
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Western blot was performed to verify the expression of prime editors in HEK293-T cells with an antibody raised against SpCas9 (mouse anti-CRISPR-Cas9; ab191468; Abcam, Cambridge, United Kingdom). HEK293-T cells transfected with PEs were lysed as described above. Samples containing 20 μg of whole protein were separated in a 10% SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) at 150 V for 75 min and transferred via semidry blotting onto nitrocellulose at 75 A for 60 min. Blocking (5% milk powder in 1 × PBS) was carried out for 1 h at RT, followed by an incubation with anti-SpCas9 (dilution: 1:1,000 in blocking buffer) overnight at 4°C and an incubation with a secondary horseradish peroxidase (HRP)-labeled anti-mouse IgG (Sigma-Aldrich, Taufkirchen, Germany) at RT for 90 min. After the addition of the HRP substrate (Amersham Bioscience, Taufkirchen, Germany) the signal was detected for 10 min on a chemiluminescence film (Amersham Bioscience, Taufkirchen, Germany).
Western blot-based verification of the BRET reporter frame correction was carried out as described above, but with a rabbit anti-RLuc antibody (Biomol, Hamburg, Germany) followed by an incubation with the anti-rabbit IgG-HRP (Sigma-Aldrich).
Western blot-based verification of the BRET reporter frame correction was carried out as described above, but with a rabbit anti-RLuc antibody (Biomol, Hamburg, Germany) followed by an incubation with the anti-rabbit IgG-HRP (Sigma-Aldrich).
5
Versican Protein Expression Analysis
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Proteins (50 mg) were separated by SDS-PAGE on 3-8% Tris-Acetate gels or 4-12% Bis-Tris gels (NuPAGE1, Invitrogen), and transferred to PVDF membranes (Amersham Biosciences). Membranes were blocked with 5% non-fat milk and incubated with rabbit anti-human versican antibody (0.2 mg/ml, A4476, Sigma-Aldrich, St Louis, MO) at 4 °C overnight. Detection was performed with a peroxidase conjugated anti-rabbit secondary antibody and ECL-substrate (ECLTM Western blotting detection system, Amersham Biosciences). Membranes were exposed to chemiluminescence film (Amersham Biosciences), which was scanned into the computer for data analysis. Quantification of immunoreactive bands was performed using ImageJ (http://rsb.info.nih.gov/ij/ ).
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