Proteins were extracted from tissues or cells in RIPA assay and protein concentration was determined by a BCA protein assays kit (Thermo Scientific). Protein were separated into 10% SDS‐PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corp.). The PVDF membranes were incubated by ORMDL3 (ab211522, 1:1000, Abcam), p‐PERK (3179, 1:1000, Cell Signaling Technology, Inc.), PERK (5683, 1:1000, Cell Signaling Technology, Inc.), p‐eiF2α (ab32157, 1:1000, Abcam), eiF2α (ab169528, 1:1000, Abcam), ATF4 (ab184909, 1:1000, Abcam), HSPA5 (ab21685, 1:1000, Abcam), GPX4 (ab125066, 1:1000, Abcam) and β‐actin (ab8226, 1:10000, Abcam) at 4°C overnight. PVDF membranes were incubated with horseradish peroxidase‐conjugated secondary antibodies (A0208, A0216, Beyotime, 1:5000) for 2 h. The signal was tested with the chemiluminescence system (Amersham Pharmacia).
Ab169528
Manufactured by Abcam
Sourced in United States
Ab169528 is a primary antibody that can be used to detect the target antigen in various applications. The core function of this product is to specifically bind to and identify the target protein in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ab8226, by Abcam (3 mentions)
Ab184909, by Abcam (3 mentions)
Ab21685, by Abcam (3 mentions)
Ab32157, by Abcam (2 mentions)
Ab229912, by Abcam (2 mentions)
Ab11419, by Abcam (2 mentions)
Ab32042, by Abcam (2 mentions)
Ab6721, by Abcam (2 mentions)
Ab62484, by Abcam (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Ab125066, by Abcam (1 mentions)
Ab211522, by Abcam (1 mentions)
Chemiluminescence system, by Cytiva (1 mentions)
Bca protein assays kit, by Thermo Fisher Scientific (1 mentions)
P perk, by Cell Signaling Technology (1 mentions)
A0216, by Beyotime (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
A0208, by Beyotime (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
8 protocols using ab169528
1
Western Blot Analysis of Cellular Proteins
2
Western Blot Analysis of ER Stress Markers
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Total cells and tissues were prepared with RIPA buffer containing proteinase inhibitor (Pierce, Rockford, IL, USA). NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) were used to separate the cytosolic fraction and nuclear extracts. Supernatant samples were separated by 10% or 15% SDS-PAGE and transferred onto nitrocellulose membranes (Millipore, Billerica, WI, USA). The membranes were probed, after blocking with 5% nonfat milk, with primary antibodies (anti-PERK, Abcam, Ab229912; anti-eIF2, Abcam, Ab169528; anti p-eIF2, Abcam, Ab32157; anti-ATF4, Proteintech, 10835-1-AP; anti-CHOP, Proteintech, 15204-1-AP; anti-β-actin, Proteintech, 66009-1-lg; anti-NF-κB, Abcam, Ab16502; and anti-H3, Proteintech, 17168-1-AP) at 4°C overnight. After washing away the unbound antibody on the second day, the membranes were washed and incubated with HRP-conjugated rabbit secondary antibody (Beyotime) at room temperature for 1 h. The signals from the immunoreactive proteins were detected using the ECL reagent (Millipore).
3
Protein Expression Analysis in Hepatocytes
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The treated human LO-2 hepatocytes were lysed to isolate proteins, which were subsequently loaded and separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, the proteins were transferred onto the polyvinylidene difluoride (PVDF) membrane and incubated with 5% nonfat milk. The membrane was then incubated with primary antibody against NOX-4 (1:1000, ab133303, Abcam), NLRP3 (1:1000, ab263899, Abcam), p-eIF-2α (1:1000, ab169528, Abcam), ATF4 (1:1000, ab184909, Abcam), GADD34 (1:1000, ab236516, Abcam), CHOP (1:1000, ab11419, Abcam), NF-κB p65 (1:1000, ab288751, Abcam) or β-actin (1:1000, ab8226, Abcam), which was further incubated with an HRP-conjugated secondary antibody. Finally, the blots were developed with electrochemiluminescence (ECL) reagents and analyzed using Image J software [25 (link)].
4
Western Blot Analysis of Protein Markers
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The proteins were extracted from cells using radio-immunoprecipitation assay lysis buffer (Beyotime, Shanghai, China). The total protein concentrations were detected with bicinchoninic acid protein assay kit (Beyotime, Shanghai, China). The same amount of proteins (20 µg) was packed into each electrophoresis tank, then separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA). Subsequently, the membranes were blocked with 10% skim milk at approximately 25°C. Then, the membranes were washed and incubated with the primary antibodies at 4°C for about 24 hour. Primary antibodies including rabbit anti-CHOP (ab194533, 1:400, Abcam, Cambridge, UK), rabbit anti-Grp78 (ab21685, 1: 500, Abcam), rabbit anti-eIF2α (ab169528, 1: 400, Abcam), rabbit anti-cleaved caspase-3 (ab32042, 1: 400, Abcam), rabbit anti- Janus kinase (JAK)2 (ab108596, 1: 300, Abcam), and rabbit anti-STAT3 (ab68153, 1: 500, Abcam) were used for incubated with goat anti-rabbit IgG H&L (HRP) (ab205718, 1:1000, Abcam) for 1 hour at room temperature. Finally, they were put into an enhanced chemiluminescence system (Bestbio, Shanghai, China) to develop the signals and scan the protein bands with Image J software. GAPDH was used as an endogenous control.
5
Protein Extraction and Analysis of Bone Tissue
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Briefly, the bone tissue was transferred into a mortar filled with liquid nitrogen and ground into powder. Then the powdered bone tissue was collected into a 1.5 mL EP tube and added with protein lysate, which was lysed at 0°C for 30 min. Then the samples were centrifuged at 4°C for 15 min at 12,000 rpm. The supernatant containing total protein was extracted and stored at −80°C. The total protein was dissolved with trypsin and quantified using a BCA Kit (Beyotime, Haimen, China). The protein samples were then applied to a 10% polyacrylamide gel for separation, and proteins were subsequently subjected onto polyvinylidene fluoride membranes (Thermo Fisher, CA, USA), followed by incubation with primary antibodies purchased from Abcam (Cambridge, MA, USA) including anti-human BMP2 (ab14933, 1:1000), anti-human ATF4 (ab184909, 1:5000), anti-human TMEM119 (ab210405, 1:1000), anti-rat BMP2 (ab214821, 1:1000), anti-rat ATF4 (ab186284, 1:500), anti-rat EIF2AK3 (ab229912, 1:1000), anti-rat EIF2A (ab169528, 1:1000) and anti-β-actin (ab8226, 1:2000) antibodies. The membranes were then incubated with the secondary antibody goat anti-rat IgG (ab150165, 1:2000) for 1 h at room temperature.14 (link) The density of blots was normalized by using β-actin as an internal reference, and captured by ImageJ software (Rawak Software Inc., Stuttgart, Germany).
6
Investigating ER Stress-Induced Apoptosis Signaling
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Cells were lysed by RIPA lysis (P0013B, Beyotime, China) supplied with PMSF and the lysates were quantitated by Bio-Rad DC Protein Assay kit (Ewell, China). The protein sample was separated using freshly-prepared SDS-PAGE, electrotransferred onto PVDF membranes, and probed with primary antibodies. After that, the membranes were re-probed with goat anti-rabbit IgG (1:10,000, ab6721, Abcam). Immunoblots were visualized with enhanced chemiluminescence detection reagents and captured under the SmartView Pro 2000 (UVCI-2100, Major Science, USA) microscope. Gray value of target protein bands was quantified using Image J software, with GAPDH used for normalization. Primary antibodies used: GRP78 (Abcam, ab21685, rabbit, 1:2000), IRE1 (Abcam, ab37073, rabbit, 1:1000), CHOP (Abcam, ab10444, rabbit, 1:1000), eIF2α (Abcam, ab169528, rabbit, 1:1000), cleaved-caspase3 (Abcam, ab32042, rabbit, 1:1000), caspase3 (Abcam, ab13847, rabbit, 1:500) and caspase 12 (Abcam, ab13847, 1:500).
7
Antibody Characterization for Protein Analysis
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Anti-CHOP antibody (ab11419), anti-p-eIF2α antibody (ab32157), anti-eIF2α antibody (ab169528), anti-XBP1 anbibody (ab37152), anti-caspase-12 antibody (ab62484), Ms mAb to NeuN (ab104224), Rb mAb to NeuN (ab177487), and anti-β-Tubulin antibody (ab179513) were purchased from abcam (Cambridge, MA, USA). Anti-ATF-4 antibody (sc-200) was purchased from Santa Cruz (Santa Cruz, CA, USA). Anti-ATF6 antibody (70B1413.1) were purchased from Novus Biological (Littleton, Co, USA). Salubrinal and GSK2606414 were purchased from TargetMol (Boston, MA, USA).
8
Immunofluorescence Assay with Apoptosis Markers
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The primary antibodies, including aFGF (ab169748), NeuN (ab177487), caspase 12 (ab62484), caspase3 (EPR18297), EIF–2α (ab169528), CHOP (ab11419), and GRP78 [EPR4041 (2)] and the secondary antibodies: goat anti-rabbit 488 (ab150077), goat anti-mouse 488 (ab150113), goat anti-rabbit (HRP) (ab6721), and rabbit anti-mouse (HRP) (ab6728) were all bought from Abcam. The DAPI is also provided by Abcam (MC, United Kingdom).
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