Cellular proteins were extracted for western blot analysis as previously described (20 (link)). The cell lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and run under standard conditions. Subsequently, the proteins were removed to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% nonfat milk at room temperature for 1 h, the PVDF membranes were incubated with diluted primary antibodies SMA (1:400 dilution; sc-53142), SM22α (1:300 dilution; sc-51442) (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), FGF9 (1:800 dilution; ab9743), PDGFRβ (1:1,000 dilution; ab32570) (both from Abcam, Cambridge, MA, USA), and antibody against GAPDH (1:1,000 dilution; sc-47724; Santa Cruz Biotechnology) at 4°C overnight. The membranes were washed, further incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies (anti-mouse and anti-rabbit, ZB-2305 and ZB-2305; both from Zhongshan Goldenbridge Biotechnology, Beijing, China) at 37°C. Subsequently, the membranes were detected with BeyoECL Plus (Beyotime Institute of Biotechnology, Haimen, China), and further analysis of protein band densitometry was facilitated by Quantity One software (Bio-Rad, Hercules, CA, USA).
Zb 2305
Manufactured by Zhongshan Golden Bridge Biotechnology
Sourced in China
The ZB-2305 is a laboratory centrifuge designed for general-purpose applications. It features a compact and durable construction and can accommodate various sample volumes and tube sizes. The centrifuge provides consistent and reliable performance to support common laboratory tasks.
Automatically generated - may contain errors
Lab products found in correlation
Zb 2301, by Zhongshan Golden Bridge Biotechnology (11 mentions)
Bca kit, by Thermo Fisher Scientific (6 mentions)
β actin, by Zhongshan Golden Bridge Biotechnology (2 mentions)
Anti pi h2axs139, by Cell Signaling Technology (2 mentions)
Ab23707, by Abcam (1 mentions)
Immobilon western chemiluminescent reagent, by Merck Group (1 mentions)
Gtx110089, by GeneTex (1 mentions)
Np 40 lysis buffer, by Beyotime (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
Phenylmethylsulfonyl fluoride, by Solarbio (1 mentions)
Ripa cell lysis buffer, by Solarbio (1 mentions)
Phosphate buffer saline (pbs), by Solarbio (1 mentions)
Gapdh sc 166545, by Santa Cruz Biotechnology (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Protease inhibitor cocktail, by Solarbio (1 mentions)
Ab9561, by Abcam (1 mentions)
Ab2721, by Abcam (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
15 protocols using zb 2305
1
Western Blot Analysis of Cellular Proteins
2
Western Blot Analysis of PINK1 and Parkin
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NP-40 Lysis Buffer (Beyotime, Shanghai, China) was used to extract total proteins as in our previous study19 (link). After extraction, the proteins were stored at −80 °C for subsequent western blot analysis. The analysis was performed as described previously57 (link). Briefly, proteins were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane, and then incubated with primary rabbit antibodies specific to PINK1 (1:1000, ab23707, polyclonal, Abcam, Cambridge, MA, UK), Parkin (1:1000, A0968, polyclonal, ABclonal, College Park, MD, USA), Hsp60 (1:5000, GTX110089, polyclonal, Genetex Inc., Irvine, CA, USA) and GAPDH (1:2000, Zhongshan Golden Bridge Biotechnology, Beijing, China) or mouse antibodies specific to COX IV (1:2000, GTX101499, polyclonal, Genetex Inc., Irvine, CA, USA) and β-actin (1:2000, Zhongshan Golden Bridge Biotechnology, Beijing, China). The samples were incubated with horseradish peroxidase (HRP) -conjugated secondary antibodies (1:20000; ZB-2305 and ZB-2301, Zhongshan Golden Bridge Biotechnology, Beijing, China), and developed with Immobilon Western Chemiluminescent reagents (Millipore, Billerica, MA, USA).
3
Protein Extraction and Western Blotting
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Cells were washed with phosphate buffer saline (PBS) (Solarbio) and resuspended with RIPA cell lysis buffer (Solarbio) containing 1% phenylmethylsulfonyl fluoride (Solarbio) and 1% protease inhibitor cocktail (Sigma-Aldrich), incubated for 30 min on ice followed by centrifuged at 4 °C, 12,000 g for 15 min to collect supernatants. BCA Protein Assay Kit (Solarbio) was used to determine protein concentration. Supernatants were mixed with 5X loading buffer (Solarbio) and incubated at 100 °C for 5 min. Samples were resolved by SDS-PAGE and transferred to a PVDF membrane. The following antibody were used: β-tubulin (Beijing Zhong Shan Goldenbridge Biotechnology #TA-10, 1:1000), SRSF1 (Santa Cruz Biotechnology #sc-33652, 1:100) and horseradish peroxidase-labeled anti-mouse IgG antibody (Beijing Zhong Shan Goldenbridge Biotechnology #ZB-2305, 1:5000).
4
Immunoblotting Technique with GAPDH, p53, and CDK1
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Antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH; sc166545, 1:3000) and the tumor protein, p53 (DO-1; sc-126, 1:1000), were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). Antibodies against cyclin-dependent kinase 1 (CDK1) (ab18, 1:1000 dilution) were purchased from Abcam (Cambridge, UK). Secondary horseradish peroxidase-conjugated antibodies against mouse immunoglobulin (Ig) G (ZB2305, 1:5000) were purchased from Zhongshan Golden Bridge Biotechnology. Co., LTD (Guangzhou, China).
5
Synthesis and Characterization of 1-CP-U
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1-CP-U, synthesized according to the reported procedure (5 ), was generously provided by Ms. Ning Qizhi, who originally synthesized the agent. The chemical structure of 1-CP-U is demonstrated in Fig. 1 . A total of 1.0 g of 1-CP-U crystal was weighed, totally dissolved it in ultrapure water facilitated by a 0.25 mol/l hydrochloric acid solution, and then the pH was adjusted to 4.0 by adding 0.25 mol/l sodium hydroxide solution. Calculating the concentration of the stock solution as 63.39 mM, it was diluted to the required concentrations in conditional medium and then stored at 4°C. The antibodies were as follows: Polyclonal rabbit anti-human Bax, MMP-2 and MMP-9, monoclonal mouse anti-human Bcl-2, (Wuhan Boster Biological Technology., Ltd.; A0315-2, BA0569, BA0573 and BM0200, respectively), and β-actin was purchased from Zhongshan Golden Bridge Biotechnology Co., Ltd., (Beijing, China; TA-09). The goat anti-mouse or anti-rabbit secondary antibodies were from Zhongshan Golden Bridge Biotechnology (ZB-2305 and ZB-2301).
6
Immunoblotting Analysis of PDLSCs
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Collection and lysis of PDLSCs was conducted using RIPA Lysis Buffer which contains 1% protease inhibitor cocktail (Solarbio). Protein concentration was determined by a BCA kit (Thermo) and a total of 30 μg of protein was used for western blot analysis. The primary antibodies against RUNX2 (CST, #12556), GDF5(Abcam, RRID: ab93855), p38 MAPK(Affinity, Cat#AF6456), phosphorylated p38 MAPK (Affinity, Cat#AF4001), JNK(Affinity, Cat#AF6318), phosphorylated JNK(Affinity, Cat#AF3318), ERK(Affinity, Cat#AF0155), phosphorylated ERK(Affinity, Cat#AF1015), and β-ACTIN (Abcam, RRID: ab8226) diluted at 1:1,000 overnight at 4°C. Three washes were done using TBST. Afterwards, incubation of the membranes was done with the anti-rabbit and anti-mouse secondary antibodies (ZB-2301 and ZB-2305, Zhongshan Golden Bridge Biotechnology, Beijing, China) which is diluted at 1:10,000 at room temperature for 1 h. Visualization of the bands was done by enhanced chemiluminescence using the Bio-Rad system for detection (ChemiDocTM MP Imaging System, USA). Intensity of the bands was measured using ImageJ. β-ACTIN internal control was used to ensure equal protein loading.
7
Quantitative Western Blot Analysis
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Western blot analysis was performed, as previously described (17 (link)). Total lysis of the cells was conducted with RIPA buffers (Thermo Fisher Scientific, Inc.) and protein concentration was determined with a bicichoninic acid protein assay kit (Thermo Fisher Scientific, Inc.). Equal quantities (50 µg) of proteins were separated and transferred onto polyvinylidine difluoride membranes. The membranes were blocked with 5% non-fat dried milk, following which the membranes were probed overnight at 4°C with the following antibodies: Rabbit monoclonal anti-Wnt3a (Ab2721 1:1,000), rabbit monoclonal anti-p-GSK-3β (Ab5558; 1:2,000), rabbit monoclonal anti-p-β-catenin (Ab9561; 1:2,000), rabbit monoclonal anti-GSK-3β (Ab12456; 1:2,000), rabbit monoclonal anti-β-catenin (Ab4176; 1:2,000) or mouse monoclonal anti-β-actin (Ab3700; 1:2,000) (all from Abcam, Cambridge, MA, USA). This was followed by incubation with either horseradish peroxidase-conjugated goat anti-rabbit (ZB-2301) or anti-mouse antibody(ZB-2305) (1:5,000; Zhongshan Golden Bridge Biotechnology, Beijing, China) for 2 h at room temperature. Immunoreactive proteins were visualized using enhanced chemiluminescence, and signal intensity was detected and quantified using Alpha Imager (Alpha Innotech Corporation, San Leandro, CA, USA).
8
Western Blotting of SIRT1 in Oocytes
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For western blotting, 120 oocytes were lysed in 2×SDS sample buffer, boiled for 5 min at 100°C and then subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane. Membrane was blocked in Tris-buffered saline (TBS) containing 5% BSA and 0.1% Tween-20 for 2 h and then incubated with rabbit anti-SIRT1 antibody (1:1000) (9475; Cell Signaling Technology, Inc.) and mouse anti-β-actin antibody (1:1000) (BE0021; Easybio Technology, Beijing). After multiple washes in TBS containing 0.1% Tween-20 and incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:1000) (ZB-2301; Zhongshan Golden Bridge Biotechnology, Beijing) and horseradish peroxidase conjugated anti-mouse IgG (1:1000) (ZB-2305; Zhongshan Golden Bridge Biotechnology, Beijing), respectively, finally, the membranes were washed 3 times in TBS containing 0.1% Tween-20 and visualized using Bio-Rad ChemiDoc XRS+.
9
Western Blot Analysis of Mouse Oocytes
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A total of 200 mouse oocytes or zygotes per sample were mixed with SDS sample buffer and boiled for 5 min at 100°C for SDS–PAGE. Western blotting was performed as described previously (Zhang et al., 2004 (link)) using the antibody dilution anti-geminin (BS7535; Bioworld, St. Louis Park, MN) at 1:100; anti-actb (TA-09; Zhongshan Golden Bridge Biotechnology, Beijing, China) at 1:1000; anti-Cdt1 (07-1383; Millipore, Darmstadt, Germany) at 1:1000; anti–Pi-Chk1S345 (2348; Cell Signaling Technology, Danvers, MA) at 1:1000; and anti–Pi-H2AXS139 (9718; Cell Signaling Technology) at 1:1000. The membranes were subsequently incubated with horseradish peroxidase–conjugated secondary antibodies (1:2000; ZB2301 and ZB2305; Zhongshan Golden Bridge Biotechnology) for 1 h at 37°C. Protein bands were detected using Thermo Supersignal West Pico chemiluminescent substrate.
10
Immunohistochemical Analysis of PES1, ERα, and ERβ
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Tissue microarray construction and IHC staining were performed as described previously48 (link). Rabbit polyclonal anti-PES1 (ab72539), mouse monoclonal anti-ERα (ab1104) and mouse monoclonal anti-ERβ (ab1103) were purchased from Abcam (Abcam, Cambridge, MA, USA). Mouse monoclonal anti-BRAFV600E (26039) was obtained from NewEast Bioscience (NewEast Bioscience, Malvern, PA, USA). These antibodies were used as primary antibodies at 1:50 dilution. Biotinylated goat-anti-rabbit and goat-anti-mouse IgG (ZB-2010 and ZB-2305, Zhongshan Golden Bridge Biotechnology, China) were used as secondary antibodies at 1:500 dilution.
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