Primary antibodies for IPF lung tissue include ATF6 (Abcam, ab37149), PERK (Abcam, ab229912), IRE1 (Abcam, ab37073), Grp78 (Abcam, ab21685), CHOP (Abcam, ab11419), α-SMA (Abcam, ab119952), and Collagen I antibody (Abcam, ab138492). For mouse LR-MSC, they include ATF6 (Abcam, ab37149), PERK (Abcam, ab229912), IRE1 (Abcam, ab37073), Grp78 (Abcam, ab21685), CHOP (Abcam, ab11419), α-SMA (Abcam, ab119952), Collagen I antibody (Abcam, ab21286), anti-Smad2/3 (Abcam, ab202445), anti-pSmad2 (Abcam, ab188334), anti-pSmad3 (Abcam, ab529035), anti-Wnt10a (Abcam, ab106522), and anti-beta Catenin (Abcam, ab32572). Anti-Lamin B1 (Abcam, ab16048) was used for loading control of nuclear protein. Anti-GAPDH (Abcam, ab8245) was used for loading control for total protein.
Ab229912
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab229912 is a multiplex ELISA kit that can simultaneously detect and quantify up to 10 different analytes in a single sample. The kit utilizes magnetic bead-based technology and a multi-well plate format to provide a high-throughput solution for multi-analyte analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ab8245, by Abcam (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Ab37149, by Abcam (2 mentions)
Ab11419, by Abcam (2 mentions)
Ab16048, by Abcam (2 mentions)
Ab184699, by Abcam (2 mentions)
Ab169528, by Abcam (2 mentions)
Ab32537, by Abcam (2 mentions)
Ab155675, by Abcam (2 mentions)
Ab21685, by Abcam (1 mentions)
Ab202445, by Abcam (1 mentions)
Ab37073, by Abcam (1 mentions)
Ab106522, by Abcam (1 mentions)
Ab21286, by Abcam (1 mentions)
Ab119952, by Abcam (1 mentions)
Ab188334, by Abcam (1 mentions)
Sc 47778, by Santa Cruz Biotechnology (1 mentions)
Ab96379, by Abcam (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
18 protocols using ab229912
1
Comprehensive Antibody Panel for IPF and LR-MSC
2
Western Blot Analysis of Protein Targets
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RIPA lysis buffer was employed to lyse cells or tumor tissues, which were then separated on SDS-PAGE gels and transferred onto the polyvinylidene fluoride (PVDF) membrane. The primary or secondary antibodies were incubated together with membranes. Antibodies of anti-WDR76 (HPA039804, Sigma-Aldrich; 1:1,000 dilution), anti-β-actin (sc-47778, Santa Cruz Biotechnology; 1:1,000 dilution), anti-Flag (A8592, Sigma-Aldrich; 1:1000 dilution), anti-HRAS (2867S, Cell Signaling Technology; 1:500 dilution), anti-p-ERK (ab229912, ABCAM; 1:500 dilution) and anti-p-MEK (ab96379, ABCAM; 1:500 dilution) were used.
3
Western Blot Analysis of Apoptosis Regulators
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H1299 and A549 were rinsed with PBS. These cells were subsequently
lysed with lysis buffer (Beyotime), enduring such conditions for 30
min at 4 °C. Following the lysis, samples were centrifuged at
high velocity, facilitating protein separation. The protein concentration
within these samples was detected via a BCA assay. Each protein sample,
consisting of 25 μg, was denatured, followed by undergoing SDS–PAGE.
They were then shifted onto polyvinylidene fluoride membranes (Millipore)
and blocked, then subjected to incubation with cIAP2 antibodies at
4 °C for 12 h, which was incubated with HRP-conjugated antibodies.
The proteins were henceforth visualized using a chemiluminescence
detection kit (Beyotime). Antibodies: anti-cIAP2 (ab32059; 1:10,000;
Abcam), anti-HSP90B1 (ab23812; 1:1,000; Abcam), anti-ERK1/2 (ab184699;
1:5,000; Abcam), and anti- PERK (ab229912; 1:1,000; Abcam).
lysed with lysis buffer (Beyotime), enduring such conditions for 30
min at 4 °C. Following the lysis, samples were centrifuged at
high velocity, facilitating protein separation. The protein concentration
within these samples was detected via a BCA assay. Each protein sample,
consisting of 25 μg, was denatured, followed by undergoing SDS–PAGE.
They were then shifted onto polyvinylidene fluoride membranes (Millipore)
and blocked, then subjected to incubation with cIAP2 antibodies at
4 °C for 12 h, which was incubated with HRP-conjugated antibodies.
The proteins were henceforth visualized using a chemiluminescence
detection kit (Beyotime). Antibodies: anti-cIAP2 (ab32059; 1:10,000;
Abcam), anti-HSP90B1 (ab23812; 1:1,000; Abcam), anti-ERK1/2 (ab184699;
1:5,000; Abcam), and anti- PERK (ab229912; 1:1,000; Abcam).
4
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).
5
Western Blot Protein Quantification
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In order to determine protein concentration before the western blotting, a bicinchoninic acid (BCA) protein assay was performed. Next, equal protein samples were prepared with 4× laemmli and lysis buffer and boiled for 5 min. Samples were isolated on a 7.5% SDS‐PAGE gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore). The membrane was probed using primary antibody overnight at 4°C. Each blot was used to probe multiple antibodies, including anti‐rabbit NR2B (Abcam, ab254356, 1:1000), anti‐rabbit pNR2B‐Tyr1472 (ThermoFisher Scientific, 38–7000, 1:1000), anti‐rabbit p‐CREB (Abcam, ab32096, 1:5000), anti‐rabbit p‐AKT (Abcam, ab8805, 1:1500), anti‐rabbit p‐KAC (Cell Signaling Technology, 4781, 1:1000), anti‐mouse p‐CAMKII (Abcam, ab171095, 1:2000), anti‐rabbit p‐ERK (Abcam, ab229912, 1:1000), anti‐rabbit p‐MSK1 (Abcam, ab278550, 1:5000), and anti‐mouse actin (Sigma, A5316, 1:100,000) served as a loading control. The membrane was then incubated with horseradish peroxidase (HRP)‐conjugated anti‐rabbit or anti‐mouse IgG secondary antibody for 1 h at room temperature. Lastly, the signal was detected using the Super Signal Chemi‐luminescent Substrate (Pierce).
6
Western Blot Analysis of Signaling Proteins
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Cells were lysed with chilled RIPA buffer (Sigma-Aldrich, MO, USA). Samples were centrifuged (12,000×g), and lysates were added to LDS sample buffer. Samples were separated via SDS-PAGE and transferred to PVDF membranes (Bio-Rad, CA, USA) that were then blocked using 5% nonfat milk, incubated overnight with primary antibodies at 4 °C overnight, and incubated with a secondary antibody (1:1000; CST, MA, USA). Protein was then detected with a chemiluminescent detection system (Bio-Rad).
Primary antibodies used in this study were specific for the following: NRG1 (ab 191139, Abcam, MA; 1:500), ErbB2 (ab 241325, Abcam; 1:1000), p-Akt (ab 241325, Abcam; 1:1000), p-ERK (ab229912, Abcam; 1:1000), p-mTOR (ab 109268, Abcam; 1:500), GAPDH (ab181603, Abcam; 1:2000), p-P65(ab 53489, Abcam; 1:1000), P65 (ab 32536, Abcam; 1:5000), LaminB (ab 16048, Abcam; 1:1000).
Primary antibodies used in this study were specific for the following: NRG1 (ab 191139, Abcam, MA; 1:500), ErbB2 (ab 241325, Abcam; 1:1000), p-Akt (ab 241325, Abcam; 1:1000), p-ERK (ab229912, Abcam; 1:1000), p-mTOR (ab 109268, Abcam; 1:500), GAPDH (ab181603, Abcam; 1:2000), p-P65(ab 53489, Abcam; 1:1000), P65 (ab 32536, Abcam; 1:5000), LaminB (ab 16048, Abcam; 1:1000).
7
Comprehensive Immunoblotting Antibody Panel
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The following primary antibodies were used: mouse monoclonal antibodies against MHV N and S proteins (kind gifts of Dr Helmut Wege, University of Würzburg), rabbit polyclonal anti-SARS-CoV-2 spike glycoprotein antibody (ab272504, Abcam) mouse anti-GAPDH (IgM specific, G8795, Sigma-Aldrich), mouse anti-Flag (F3165, Sigma-Aldrich), rabbit anti-HA (3724, Cell Signaling Technology), rabbit anti-PERK (ab229912, Abcam), rabbit anti-HERPUD1 (ab150424, Abcam), rabbit anti-GRP78 (BIP, ab108613, Abcam), rabbit anti-eIF2α (9722, Cell Signaling Technology), rabbit anti-phospho-eIF2α (Ser51, 9721, Cell Signaling Technology), rabbit anti-ATF4 (10835-1-AP, Proteintech), rabbit anti-ATF6 (ab203119 and ab37149, Abcam), mouse anti-S6 (2317, Cell Signaling Technology) and rabbit RPL10a (ab174318, Abcam). Secondary antibodies used for western blotting were purchased from Licor: IRDye 800CW Donkey Anti-Mouse IgG (H+L), IRDye 800CW Donkey Anti-Rabbit IgG (H+L), IRDye 680RD Goat Anti-Mouse IgG (H+L) and IRDye 680RD Goat Anti-Mouse IgM (μ chain specific).
8
Western Blot Analysis of Signaling Proteins
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SW579 cells were lysed with radioimmunoprecipitation assay (Beyotime) lysate, and the protein concentration was determined using the bicinchoninic acid protein detection kits (Beyotime). The 10% sodium dodecyl sulfate‐polyacrylamide (SDS‐PAGE) gel was prepared, and then the protein was separated in SDS‐PAGE gel and electrotransferred to polyvinylidene fluoride (PVDF) membranes (Millipore). Tris‐buffered saline tween (TBST) was used to prepare 5% skim milk, and then PVDF membranes were put into the milk, shaken, and blocked at room temperature for 1 h to block the nonspecific binding. The membranes were added with primary anti‐KIT (ab283653; 1:1000; Abcam), anti‐p‐extracellular signal‐regulated kinases (ERK) (ab229912; 1:1000; Abcam), anti‐ERK (ab32537; 1:1000; Abcam), anti‐PD‐L1 (ab205921; 1:100; Abcam), anti‐GAPDH (ab8245; 1:5000; Abcam), and incubated overnight at 4°C. The membranes were washed with TBST twice and incubated with horseradish peroxidase‐labeled goat anti‐rabbit IgG (ab48386; 1:2000; Abcam) at room temperature for 1 h. Next, the membranes were developed with enhanced chemiluminescence working fluid (Millipore) and photographed. ImageJ software (version 1.48; NIH) was adopted to detect protein band density, with GAPDH as an internal reference.
9
Immunohistochemical Analysis of Immune Markers
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Paraffin-embedded blocks were cut into 4-μm thick sections. The dewaxed and hydrated tissue sections were incubated with anti-PU.1 (Abcam, ab88082), anti-CD23 (Abcam, ab254162), anti-p-ERK (Abcam, ab229912), anti-CCL20 (Abcam, ab9829) and anti-IL-8 (Abcam, ab18672) antibodies for 2 h at room temperature and subsequently incubated with a goat-anti-rabbit antibody for 40 min. The degree of staining was determined with diaminobenzidine (DAB) chromogen (Bio-Rad, Inc., CA, USA) and detected with a microscope (Olympus, Japan).
10
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).
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