Anti-α-SMA (cs19245), anti-p-AMPKα (cs2535), anti-caspase-1 (cs24232) and anti-NOD-like receptor thermal protein domain associated protein 3 (NLRP3) (cs15101) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-CollagenI (ab34710), anti-Tissue Inhibitors of Metalloproteinase 1 (TIMP1) (ab61224), anti-caspase-3 (ab179517), anti-beclin-1 (ab210498), anti-Bcl-2 (ab194583) antibodies and the horseradish peroxidase (HRP)-conjugated goat anti-rabbit (ab97051) were purchased from Abcam (Cambridge, MA, USA). Anti-CollagenI (14695-1-AP), anti-SIRT3 (10099-1-AP), anti-F4/80 (28463-1-AP), anti-caspase-6 (10198-1-AP), anti-caspase-9 (10380-1-AP) and anti-GAPDH (60004-1-Ig) antibodies were purchased from Proteintech (Wuhan, HubChina). Anti-Matrix metalloproteinase-13 (MMP-13) (sc515284) and anti-Interleukin (IL)-1R1 (sc393998) antibodies were purchased from Santa Cruz Biotechnology (Stanta Cruze, CA, USA). Anti-IL-1β (mab4012) antibodies were purchased from R&D Systems (Minneapolis, MN, USA). Alexa Fluor 488 Goat anti-Mouse IgG (A11034) and Alexa Fluor Plus 647 Goat anti-Rabbit IgG (A32727) were purchased from Beyotime Biotechnology (Shanghai, China).
Ab210498
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab210498 is a laboratory equipment product. It serves a core function related to scientific research and analysis. No further details about its intended use or capabilities can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Ab192890, by Abcam (13 mentions)
Ab56416, by Abcam (9 mentions)
Ab109012, by Abcam (8 mentions)
Ab205718, by Abcam (6 mentions)
Ab9485, by Abcam (5 mentions)
Ab128025, by Abcam (5 mentions)
Ab8245, by Abcam (5 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
Ab51520, by Abcam (4 mentions)
Ab181602, by Abcam (4 mentions)
Ab8226, by Abcam (3 mentions)
Ecl kit, by Merck Group (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Ab179517, by Abcam (2 mentions)
Ab32042, by Abcam (2 mentions)
Ab39012, by Abcam (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Ab182733, by Abcam (2 mentions)
Ma5 11757, by Thermo Fisher Scientific (2 mentions)
Ab91526, by Abcam (2 mentions)
43 protocols using ab210498
1
Antibody Resource for Cellular Analysis
2
Quantitative Protein Expression Analysis
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Total protein from tissues and cells was extracted using radio-immunoprecipitation assay lysis buffer containing phenylmethylsulfonyl fluoride, followed by lysing on ice for 30 min to collect cells. Cells were centrifuged at 8000 RPM at 4°C for 10 min to obtain supernatant. Protein concentration was detected using bicinchoninic acid protein assay kit (P0012, Beyotime, Shanghai, China). Subsequently, 50 µg of isolated proteins were dissolved in 2 × SDS loading buffer and boiled at 100°C for 10 min. Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA). Membranes were blocked with 5% skimmed milk for 1 h, probed with diluted primary antibodies to BNIP3 (1:1000, #44,060, CST, MA), LC3 (1:5000, PA1-46,286, Sigma, MO), BECN1 (1:1000, ab210498, Abcam, Cambridge, UK), BCL-2 (1:1000, #15,071, CST, MA) and GAPDH (1:5000, #2118, CST, MA) overnight at 4°C, and further re-probed with the secondary goat anti-rabbit (1:20,000, ab205718) or anti-mouse (1:20,000, ab205719) immunoglobulin G (IgG) antibodies (Abcam, Cambridge, UK) for 1 h at room temperature. Electrogenerated chemiluminescence (ECL; WBKLS0100, Millipore, MA) was applied for visualization. Gray scale value of protein bands was quantified by ImageJ software (NIH).
3
Immunohistochemical Analysis of Liver Fibrosis
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The liver tissues were fixed with buffered formalin (10%), embedded in paraffin, and cut into 4 µm-thick sections. The slices were incubated with citrate buffer (pH 6.0) for 5 min at 108 °C, pretreated with 3% hydrogen peroxide (H2O2) for 15 min at room temperature and washed with PBS. Then, the slices were incubated with normal goat serum for 20 min, followed by incubation with primary antibody against α-SMA (1:500, ab108424, Abcam), Col1α1 (1:50, PA5-36227, Invitrogen) and BECN1 (1:100, ab210498, Abcam) overnight at 4 °C. Besides, the slices were incubated with secondary antibody (1:500, horseradish peroxidase-conjugated anti-rabbit IgG) and the reaction products were visualized with DAB solution.
4
Protein Analysis in BC Tissue Samples
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Proteins were extracted from BC tissue samples and cells using RIPA reagent, and quantified using a bicinchoninic acid (BCA) protein quantitation kit. Then, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed for protein separation, after which the proteins were moved onto polyvinylidene fluoride membranes. After being sealed with 5% defatted milk, the membranes were incubated with primary antibodies and then with horseradish peroxidase-conjugated secondary antibody. The following antibodies were used, all of which were bought from Abcam (Shanghai, China): PRRX1 (1:1,000, ab106834; Abcam), LC3B (1:3,000, ab63817; Abcam, containing LC3-I and LC3-II), Beclin-1 (1:1,000, ab210498; Abcam), FOXM1 (1:1,000, ab245309; Abcam), and β-actin (1:1,000, ab8226; Abcam). An enhanced electrochemiluminescence (ECL) detection system (Millipore) was used to detect the protein bands.
5
Apoptosis and Autophagy Protein Analysis
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Cell proteins extraction was done using RIPA lysis buffer (Biotechnology, Shanghai, China) conjugated with protease inhibitor cocktail (Beyotechnology, Shanghai, China). Proteins were loaded into 12% SDS-PAGE gel. After electrophoresis, proteins were transferred onto polyvinylidene fluoride membrane (Thermo Fischer Scientific, USA). These membranes were blocked with 4% bovine serum albumin for 1 h at room temperature, after which they were incubated with primary antibodies at 4 o C for about 24 h. Next, these membranes were incubated with the appropriate secondary antibodies at room temperature for around 1 h. The primary antibodies against cleaved caspase-3 (1:1000, Abcam, UK; ab32042), cleaved caspase-9 (1:1000, Abcam, UK; ab2324), ATG2B (1:1000, Abcam, UK; ab189934), BECN1 (1:1000, Abcam, UK; ab210498), LC3B (1:1000, Abcam, UK; ab192890), cyclin D1 (1:1000, Abcam, UK; ab16663) were used in this research. GAPDH (1:1000, Beyotechnology, China; AF1186) was used as the internal control. The membranes were incubated for 1 h with HRP-conjugated secondary antibodies (Santa Cruz). Finally, protein bands were visualized by ECL kits (Millipore, USA), and analyzed using ImageJ software.
6
Western Blot Analysis of Autophagy Markers
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Total proteins from tissues or cell lines were extracted using the RIPA lysis buffer (Beyotime, Shanghai, China). 20 μg proteins were separated in 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Roche, Mannheim, Germany), then the transfected membranes were blocked in Tris-buffered saline containing 5% skimmed milk powder for 2 h. Next, the membranes were incubated with Anti-SORT1 (1 μg/ml, ab16640, abcam), Anti-Beclin-1 (1:1000, ab210498, abcam), Anti-LC3B (1:2000, ab192890, abcam) or Anti-p62 (1:1000, ab56416, abcam) at 4 °C overnight. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. Protein signals was visualized by ECL chemiluminescent reagent (Millipore, MA, USA).
7
Quantification of Autophagy-Related Proteins
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Radio-immunoprecipitation assay (RIPA) buffer was used to lyse cells, and the collected protein samples were then separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. Non-specific protein binding was blocked for 1 h via the application of a 5% milk solution, followed by incubation with primary antibodies specific for Beclin1 (1:2,000, ab210498, Abcam, MA, USA), p62 (1:1,500, ab109012, Abcam), and GAPDH (1:10,000, ab9485, Abcam). Blots were then probed with secondary horseradish peroxidase (HRP)-conjugated antibodies (1:20,000, ab205718, Abcam), and protein bands were detected with an enhanced chemiluminescence (ECL) kit (Millipore, Bedford, MA, USA).
8
Molecular Mechanisms of Estrogen-Mediated Autophagy
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Estradiol (E2758, purity ≥ 98%), MPP dihydrochloride hydrate (M7068, purity ≥ 97%), PHTPP (SML1355, purity ≥ 98%) and 3-Methyladenine (3-MA, M9281, purity ≥ 99%) were purchased from Sigma (Saint Louis, USA). Antibody for NLRP3 (19771-1-AP) and IL-18 (10663-1-AP) were purchased from Proteintech (Wuhan, China). Antibody for caspase-1 (48847) was purchased from SAB signalway antibody (Maryland, USA). Antibody for GSDMD (A18281) and IL-1β (A16288) were purchased from ABclonal (Wuhan, China). Antibody for Beclin 1 (ab210498), LC3B (ab192890), SQSTM1 (ab56416), Hcy (ab15154), estrogen receptor alpha (ERα) (ab32063), estrogen receptor beta (ERβ) (ab3576), GSDMD (ab219800), and β-actin (ab8226) were purchased from Abcam (Cambridge, UK). FITC - goat Anti-mouse IgG (ab6785), TRITC - goat anti-rabbit IgG (BS10250), FITC - goat anti-rabbit IgG (BS10950), HRP - goat anti-rabbit IgG (BS13278), and HRP - goat anti-mouse IgG (BS12478) were purchased from Bioworld (Bloomington, USA). The prestained protein MW marker (26617) were purchased from Thermo scientific. Control siRNA and ERα siRNA (sc-29305) were purchased from Santa Cruz (Santa Cruz, USA).
9
Western Blot Analysis of Autophagy Markers
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Radioimmunoprecipitation assay (RIPA) buffer was used for protein extraction. The supernatants from cell lysates were run on 10% acrylamide gels by SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (Millipore). Antibodies against ROCK2 (1:1000, #ab71598, Abcam), LC3 (1:1000, #12741, Cell Signaling Technology), p62 (1:1000, #ab56416, Abcam), and Beclin1 (1:1000, #ab210498, Abcam) and an HRP-conjugated secondary antibody (1:2000) were employed for western blotting. A chemiluminescence western blotting detection system (Bio-Rad) was used for protein detection.
10
Western Blot Analysis of Autophagy and GFRA1 in Osteosarcoma
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Osteosarcoma cells were lysed with RIPA lysis solution (Beyotime, Shanghai, China). Proteins extracted from osteosarcoma cells were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were blocked with nonfat milk for 1 h at room temperature and then incubated at 4°C overnight with the following primary antibodies: anti-LC3 (1:5000, ab51520, Abcam), anti-Beclin-1 (1:8000, ab210498, Abcam), anti-p62 (1:5000, ab155686, Abcam), anti-GFRA1 (1:5000, ab84106, Abcam), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:20000, ab37168, Abcam). Afterward, the membranes were incubated with the horseradish peroxidase-conjugated secondary antibody (1:5000, ab205718, Abcam) for 2 h at room temperature. Protein bands were detected through the enhanced chemiluminescence system (Beyotime).
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