Total protein was extracted from cells or tissues using RIPA buffer (Beyotime) and quantified with an Easy II Protein Quantitative Kit (TransGen Biotech). The protein samples were subjected to SDS‐PAGE and then transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk for 1 hour and incubated with primary antibodies against PARP1 (1:1000, PB9309, Boster Biological Technology), HMGB1 (1:1000, A00066‐1, Boster Biological Technology), Bcl‐2 (1:1000, A00040‐2, Boster Biological Technology), Bax (1:500, bs‐0127R, Bioss, Beijing, China), cleaved caspase‐3 (c‐Caspase3; 1:500, BA3257, Boster Biological Technology), acetylated lysine (1:500, MA1‐2021, Thermo Fisher), cytochrome C (1:500, bs‐0013R, Bioss), COX IV (1:2000, bsm‐52750R, Bioss) and GAPDH (1:1000, BA2913, Boster Biological Technology) overnight at 4°C. Then, HRP‐conjugated AffiniPure Goat Anti‐rabbit/mouse IgG secondary antibody (1:5000, Boster Biological Technology) was incubated for 1 hour. The EasySee® Western Blot Kit (TransGen Biotech) was used to detect the protein bands.
Bs 0013r
Manufactured by Bioss Antibodies
Sourced in China, Switzerland
The Bs-0013R is a laboratory equipment product designed for general research applications. It functions as a centrifuge, providing a controlled environment for separating and isolating various components from liquid samples through centrifugal force. The technical specifications and detailed capabilities of this product are not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Easy 2 protein quantitative kit, by Transgene (1 mentions)
Bs 0127r, by Bioss Antibodies (1 mentions)
Ba3257, by Boster Bio (1 mentions)
Ma1 2021, by Thermo Fisher Scientific (1 mentions)
Ba2913, by Boster Bio (1 mentions)
Easysee western blot kit, by Transgene (1 mentions)
A00066 1, by Boster Bio (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Mito tracker green, by Beyotime (1 mentions)
Bs 20773r, by Bioss Antibodies (1 mentions)
Ripa p1003b, by Beyotime (1 mentions)
Hatf00010, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Sa00001 1, by Proteintech (1 mentions)
Sa00001 2, by Proteintech (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
4 protocols using bs 0013r
1
Protein Extraction and Western Blot Analysis
2
Cytochrome C distribution in HCM cells
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The distribution of cytochrome C in HCM cells was observed by immunofluorescence staining. After fixation with 4% paraformaldehyde, the HCM cells were permeabilized with Triton X‐100 and blocked with normal goat serum. Then, HCM cells were incubated with primary antibody cytochrome C (1:100, bs‐0013R, Bioss) at 4°C overnight. Next, Mouse Anti‐Rabbit IgM/Cy3 (1:100, bs‐0369M‐Cy3, Bioss) was incubated for 1 hour. After nuclear staining with DAPI and mitochondrion labelling with mito‐Tracker Green (Beyotime), the fluorescence was observed under a fluorescent microscope.
3
Protein Analysis of H9c2 Cells Treated with SXD
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H/R-induced H9c2 cells were treated with SXD, and then the proteins from each treatment group were isolated by radio immunoprecipitation assay buffer (RIPA, P1003B, Beyotime, China). The supernatant was centrifuged at 14,000 rpm for 15 min at 4 °C, and the total protein concentration was determined, followed by quantitation with the bicinchoninic acid assay (BCA) protein assay kit (P0010, Beyotime, China). After denaturation in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, the equal amounts of proteins were transferred to nitrocellulose membrane (HATF00010, Millipore, USA). Blots were blocked with 5 % fat-free milk in TBS with Tween-20 (TBST) at room temperature (approximately 25 °C) for 1 h, and then Caspase3 (1:500, 19677-1-AP, Proteintech, USA), Caspase9 (1:500, bs-20773R, Bioss, Switzerland), Cytochrome C (1:500, bs-0013R, Bioss, Switzerland), β-actin (1:5000, 200068-8F10, ZEN-BIOSCIENCE, China) primary antibodies were added in 5 % blocking buffer at 4 °C overnight. After incubation with the corresponding HRP-conjugated secondary antibodies (1:2000, SA00001-1 and SA00001-2, Proteintech, USA), the enhanced chemiluminescence (ECL) kit (WBKLS0500, Millipore, USA) was used for detection. The grey scale values of protein bands were analyzed using Image J software (NIH, USA).
4
Evaluation of apoptosis-related proteins
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Briefly, cells were seeded in 6-well plates and treated with 17 . After 3 h, cells were irradiated with the laser light (660 nm, 50 mW, 5 J/cm2) and incubated for an additional 24 h. Total protein was isolated from the AsPC-1, MIA-PaCa-2, and PANC-1 cells by using the RIPA lysis buffer (150 mM sodium chloride, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris adjusted to pH 8.0) containing 1X proteases and phosphatases inhibitors for 1 h. The lysates were centrifuged at 10,000× g for 10 min at 4 °C, and the supernatant fractions were collected. Equal amounts of proteins from the samples were separated using 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred in Immobilon P membranes (Millipore Corp., Bedford, MA, USA). The membranes were incubated at 4 °C overnight with different primary antibodies: anti-mouse anti-Bcl-2 (Bioss, bs-52022M), anti-rabbit β-Actin (abcam, 8226) antibodies, and cytochrome c (Bioss, bs-0013R). The results were visualized using an enhanced chemiluminescence (ECL) reagent and the images were taken using a luminescent image analyzer (Amersham, GE Healthcare, Piscataway, NJ, USA).
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