The complete protein was extracted and quantified using the BCA method. We used the SDS-PAGE to collect 40 µg of total protein and transferred it to a PVDF membrane (GE Healthcare). Therefore, the membrane sealing, and antibody incubation were conducted with 5% skimmed milk powder at room temperature for 1 h. Subsequently, the antibodies were diluted into the blocking solution following the manuscript instructions and incubated with the membrane at 5 ℃ overnight. The primary antibodies included β—actin first antibody (beyotime), SLPI first antibody (Abcam, ab17157), PUMA first antibody (Abcam, ab9643) and p-foxo3 α first antibody (Abcam, ab9643), Ab47285) FoxO3 α first antibody (Abcam, ab12162) p65 (Abcam, ab16502) p-p65 (Abcam, ab76302) p-Akt (Abcam, ab81283) Akt (Abcam, ab8805) Bax (Abcam, ab32503) c-caspase3 (Abcam, ab2302) Ki67 (Abcam, ab15580). Furthermore, we used the TBST to wash the membrane incubated with a primary antibody three times, 10 min each time. Then, secondary antibody second antibody (beyotime) was diluted and incubated with the membrane at room temperature for 2 h, washed with TBST three times, 10 min each time before the bands' observation through Amersham ECL solution, and assessed with Multi gauge computer software (Berthold, Bundoora, Australia).
Ab9643
Manufactured by Abcam
Sourced in United Kingdom
Ab9643 is an antibody product offered by Abcam. It is a laboratory reagent intended for use in research applications. The core function of this product is to detect the presence of a specific target molecule, as per the manufacturer's specifications. Further details about the intended use or application of this product are not provided.
Automatically generated - may contain errors
Lab products found in correlation
Ab32503, by Abcam (3 mentions)
Ab8805, by Abcam (3 mentions)
Ab2302, by Abcam (2 mentions)
Ab17157, by Abcam (1 mentions)
Ab15580, by Abcam (1 mentions)
Multigauge computer software, by Berthold Technologies (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Ab76302, by Abcam (1 mentions)
Ab16502, by Abcam (1 mentions)
Ab81283, by Abcam (1 mentions)
Ab12162, by Abcam (1 mentions)
Amersham ecl, by Cytiva (1 mentions)
Gapdh, by Abcam (1 mentions)
Bca protein assay kit, by CoWin Biotech (1 mentions)
Pvdf microporous membrane, by Merck Group (1 mentions)
Protease inhibitor, by Applygen (1 mentions)
Ab124956, by Abcam (1 mentions)
Ab1101, by Abcam (1 mentions)
Ab33186, by Abcam (1 mentions)
Ab66705, by Abcam (1 mentions)
13 protocols using ab9643
1
Protein Quantification and Western Blot Analysis
2
Investigating miR-373 Regulation in MG-63 Cells
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Standard Western blotting was conducted for protein expression assays from MG-63 cells with miR-373 mimic, inhibitor, and miR-control. Briefly, proteins were isolated with RIPA lysis buffer containing 1 mg of protease inhibitors (Applygen Technologies Inc., Beijing, P.R. China) after 2 days of transfection. The protein content was quantified using Bicinchoninic Acid (BCA) Protein Assay Kit (CoWin Biotech Co., Ltd., Beijing, P.R. China). The following primary antibodies p53 (ab1101), p21 (ab109520), p53 upregulated modulator of apoptosis (Puma; ab9643), B-cell lymphoma-2 associated X (Bax; ab32503), plasminogen activator inhibitor (PAI; ab66705), PI3K (ab86714), AKT (ab8805), Rac1; ab33186), c-Jun N-terminal kinase (JNK; ab124956), and the internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were from Abcam (Cambridge, UK). Subsequently, secondary antibodies were marked by horseradish peroxidase for 2 h at 37°C. Samples were then electrotransferred to polyvinylidene fluoride (PVDF) microporous membrane (Millipore, Boston, MA, USA). The bands were visualized by the Odyssey CLx equipment (LI-COR Bioscences, Lincoln, NE, USA).
3
Renal Protein Extraction and Western Blot
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Proteins were extracted from renal tissue using an ice-cold lysis buffer, and the protein concentrations were quantified using a BCA assay kit (PC0020; Solarbio). SDS-PAGE was used to separate equal amounts of protein. The blocked membrane was incubated using anti-Bax (1 : 500; WL01637, WanleiBio), anti-P-p53 (1 : 2,000; ab1431, Abcam), anti-PUMA (1 : 3,000; ab9643, Abcam), anti-caspase-3 (1 : 2,000; 9661, CST), anti-Nrf2 (1 : 500; ab89443, Abcam), anti-HO-1 (1 : 500; WL01637, WanleiBio), anti-OCT2 (1 : 500; ab243153, Abcam), anti-MRP2 (1 : 500; ab203397, Abcam), and anti-β-actin (1 : 1,000; WL01372, WanleiBio) antibodies overnight at 4°C. Then, the membranes were incubated with a suitable horseradish peroxidase-conjugated secondary antibody for 60 min at room temperature. Finally, an ECL western blotting substrate was used to visualize the immunoblots.
4
Protein Extraction and Western Blot Analysis
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Cells were collected and lysed using a mammalian protein extraction reagent containing a protease inhibitor cocktail. After centrifugation at 17 000 g for 15 min, the protein content in the supernatant was determined using the BCA protein assay kit (Bio-Rad, Shanghai, China). Whole Cell Lysate was used for the assay. Antibodies against pyruvate kinase M2 (PKM2), PUMA (ab9643), RIP3 (ab56164), p-RIP3 (S227, ab209384), p-MLKL (S358, ab187091), Bax (ab32503), Bcl-2 (ab32124), Bid (ab32060), Bcl-XL (ab32370) were purchased from Abcam (Cambridge, MA, USA), antibodies against β-Actin (sc-47778) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
5
Western Blot Immunodetection of Apoptosis Markers
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Cell lysates were heat denatured, separated by SDS‒PAGE, transferred to PVDF membranes (abs931, Absin, Shanghai, China), and blocked with 5% skimmed milk. Then, membranes were incubated, respectively with anti-BRAF (1:1000, ab33899), anti-GPX4 (1:1000, ab125066), anti-PUMA (1:1000, ab9643), anti-LC3B (1:2000, ab192890), anti-MLKL (1:500, ab184718), anti-phospho-MLKL (1:1000, ab196436), anti-ERK1/2 (1:1000, ab184699), anti-Ubiquitin (1:1000, ab134953), anti-AMPKα (1:2000, ab32047), anti-p62 (1:1000, ab109012), anti-Beclin1 (1:2000, ab207612) primary antibodies from Abcam (UK), anti-Alpha Tubulin (1:1000, 11224-1-AP), anti-FOXO3a (1:1000, 10849-1-AP), anti- SLC7A11 (1:1000, 26864-1-AP), ati-H3 (1:1000, 17168-1-AP) primary antibodies from Proteintech (IL, USA), anti-Cleaved Caspase-3 (1:1000, #9661), anti-phospho-Beclin1 (1:1000, #14717), anti-phosphor-AMPKα (1:1000, #2535), anti-phospho-FOXO3a (1:1000, #64616), anti-phospho-ERK1/2 (1:1000, #4370) primary antibodies from Cell Signaling Technology (MA, USA) and anti-ATG5 (1:1000, ET1611-38) from HUABIO (Hangzhou, China) overnight at 4 °C and anti-rabbit (1:5000, ab205718, absin, Shanghai, China) or anti-mouse (1:5000, abs20039, absin, Shanghai, China) secondary antibodies for 1 h at room temperature. Immunoblots were visualized using an ECL detection reagent (BMU101-CN, Abbkine Scientific, Wuhan, China).
6
Western Blotting for Brain Protein Analysis
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For western blotting, mice were anesthetized, decapitated and brains were removed quickly. Crude protein was extracted by homogenizing 0.2 mg cerebral cortex in 200 µl RIPA lysis buffer containing 2 µl 1× complete protease inhibitor mixture. Lysates were centrifuged at 12,000 rpm at 4 °C for 10 min, then the supernatants were transferred to new tubes added with 1× loading buffer, and heated for 5 min at 95 °C. Protein was separated by SDS-PAGE electrophoresis and then transferred to nitrocellulose membrane at 200 mA for 2 h in 4 °C. The membrane was blocked for 2 h in 5% non-fat dry milk and then incubated with primary antibodies: rabbit anti-ARGLU1 (1:500; HPA034962, Sigma-Aldrich), rabbit anti-p21 (1:1000; cell signaling, 2947 T), rabbit anti-PUMA (1:1000; Abcam, ab9643), rabbit anti-NOXA (1:1000; Abcam, ab131088), rabbit anti-p53 (1:1000; LSBio, LS-C334397), mouse anti-p53 (1;1000; Abcam ab26), mouse anti-MDM2 (1:200; Santa Cruz sc-965), mouse anti-MDM4 (1:200; Santa Cruz sc-74468), and anti-β-actin (1:5000; Millipore), followed by incubation with HRP-conjugated secondary antibody. ECL detection kit (Bio-Rad) was used for signal detection. Data were analyzed using ImageJ (NIH)-Fuji software.
7
Protein Extraction and Western Blotting
8
Western Blot Analysis of Apoptosis Regulators in Tumor Samples
9
Immunoblotting Assay for Protein Quantification
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The cell and tissue protein were prepared with 1% NP-40 cell lysis buffer (50 mM Tris HCl [pH 8.0], 120 mM NaCl, 1% NP-40) containing 1 mM dithiothreitol, and phosphatase inhibitor cocktails I and II (Sigma). The protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis for further analysis. The following lists the primary antibody information: anti-SKP2 (H435, Santa Cruz Biotechnology), anti-p53 (FL-393, Santa Cruz Biotechnology), anti-cleaved caspase 3 (ab2302, Abcam, Cambridge, MA), anticytochrome C (ab13575, Abcam), anti-Bax (N-20, Santa Cruz Biotechnology), anti–cleaved poly(ADP-ribose) polymerase (PARP; 9545, Cell Signaling Technology, Beverly, MA), antiactin (I-19, Santa Cruz Biotechnology), anti-Flag (F7425, Sigma-Aldrich), anti–c-Myc (9E10, Santa Cruz Biotechnology), anti-p21 (ab18209, Abcam), anti-Puma (ab9643, Abcam), and anti-Mdm2 (SMP14, Santa Cruz Biotechnology). The quantified relative ratios of remaining SKP2 were obtained by densitometry using Image J software, and the data at 0 hour was set to 1.
10
Protein Expression Analysis in Cells
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Total protein of cells in each group was extracted using ProteoPrep® Total Extraction Sample Kit (Sigma, USA). Concentrations of proteins were tested by Bradford assay (Bio-Rad, USA), and protein samples were separated on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted to polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, USA). After the blockage with 5% nonfat dry milk for 1 hr at 37°C, membranes were incubated with specific primary antibodies for Puma (Abcam, ab9643, 1:1000), Bax (Abcam, ab53154, 1:1000), Bcl-2 (Abcam ab59348, 1:1000), Cleaved caspase 9 (Abcam, ab2324, 1:1000), Cyclin D1 (Abcam, ab226977, 1:1000), and CDK4 (Abcam, ab137675, 1:2000), PI3K (Abcam, ab133595, 1:2000), AKT (Abcam, ab8805, 1:500), phosphorylated (p)-PI3K (Abcam, ab182651, 1:1000), p-Akt (Abcam, ab38449, 1:1000) and GAPDH (Abcam, ab9485, 1:2000) respectively, overnight at 4°C. Then, they were treated with HRP (horseradish peroxidase)-conjugated secondary antibodies (Abcam, USA) at 37°C for 1 hr and exposed to X-ray film. Finally, immunoreactive bands were detected using enhanced chemiluminescence (ECL) detection reagents (Amersham, Arlington Heights, IL, USA). Finally, band densities were quantified by densitometry Bio-Rad ChemiDoc™ XRS+ System with Image Lab™ Software version 4.1 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
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