Primary antibodies were rabbit polyclonal anti-HMGA2 (Cell Signalling, 1:1000), rabbit monoclonal anti-HMGA1 (1:1000; Cell Signaling), and mouse monoclonal anti-FLAG M2 antibodies (1:1000; Sigma Aldrich). Secondary antibodies were horseradish peroxidase-conjugated goat anti-rabbit antibodies (1:10 000; Santa Cruz) and polyclonal goat anti-mouse immunoglobulins (1:10 000; Dako).
Horseradish peroxidase conjugated goat anti rabbit antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Horseradish peroxidase-conjugated goat anti-rabbit antibody is a secondary antibody used in various immunodetection techniques. It binds to primary rabbit antibodies and enables detection through the enzymatic activity of the conjugated horseradish peroxidase.
Automatically generated - may contain errors
Lab products found in correlation
Novex nupage electrophoresis system, by Thermo Fisher Scientific (3 mentions)
Chemiluminescence system, by Cytiva (3 mentions)
Jnk ab, by Santa Cruz Biotechnology (2 mentions)
P jnk ab, by Santa Cruz Biotechnology (2 mentions)
P38 ab, by Santa Cruz Biotechnology (2 mentions)
P p38 ab, by Santa Cruz Biotechnology (2 mentions)
P65 ab, by GeneTex (2 mentions)
Microbca kit, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence reagent, by GE Healthcare (2 mentions)
Polyclonal goat anti mouse immunoglobulin, by Agilent Technologies (1 mentions)
Anti hmga2, by Cell Signaling Technology (1 mentions)
Asc ab, by Santa Cruz Biotechnology (1 mentions)
Mmp 9 ab, by Abcam (1 mentions)
Anti tm4sf1, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti β actin, by Abcam (1 mentions)
Mini protean tgx system precast protein gels, by Bio-Rad (1 mentions)
Westernbright quantum hrp substrate, by Advansta (1 mentions)
Trans blot turbo mini pvdf transfer pack, by Bio-Rad (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
19 protocols using horseradish peroxidase conjugated goat anti rabbit antibody
1
Antibody Immunoblotting Procedure
2
Western Blot Analysis of Inflammatory Signaling
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Cells were lysed in PBS containing 1% nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 5 μg/mL aprotinin, 100 μg/mL phenylmethylsulfonyl fluoride, 1 μg/mL pepstatin A, and 1 mM ethylenediaminetetraacetic acid (EDTA) at 4 °C for 20 min. Total lysates were quantified using a microBCA kit (Thermo Fisher Scientific, Waltham, MA, USA). Proteins (10 μg) were resolved by SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to a PVDF (poly(vinylidene fluoride)) membrane. The membrane was blocked in 5% fat-free milk in PBST (PBS with 0.05% Tween-20), followed by incubation overnight with the following primary antibodies diluted in PBST: JNK Ab, p-JNK Ab, p38 Ab, p-p38 Ab, ASC Ab, procaspase-1 Ab (diluted to 1:1000, all from Santa Cruz Biotech (Dallas, TX, USA)), p65 Ab (Genetex, Taiwan) and NLRP3 Ab (Adipogen, San Diego, CA, USA). The primary antibodies were removed, and the membrane was washed extensively in PBST. Subsequent incubation with horseradish peroxidase-conjugated goat anti-rabbit antibodies (1:20,000, Santa Cruz Biotech) was performed at room temperature for 2 h. The membrane was washed extensively in PBST to remove any excess secondary antibodies, and the blot was visualized with enhanced chemiluminescence reagent (GE Healthcare, South Jakarta, Indonesia).
3
Protein Expression Analysis by Western Blot
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Cells were lysed in PBS containing 1% nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 5 μg/mL aprotinin, 100 μg/mL phenylmethylsulfonyl fluoride, 1 μg/mL pepstatin A, and 1 mM ethylenediaminetetraacetic acid (EDTA) at 4°C for 20 minutes. Total lysates were quantified using a microBCA kit (Thermo Fisher Scientific, Waltham, MA). Proteins (10 μg) were resolved by SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to a PVDF membrane. The membrane was blocked in 5% fat-free milk in PBST buffer (PBS with 0.05% Tween-20) followed by incubation overnight with the following primary antibodies diluted in PBST buffer: TM antibody (Ab), MMP-2 Ab, JNK Ab, pJNK Ab, p38 Ab, pp38 Ab, c-jun Ab, c-fos Ab, lamin B1 Ab (diluted used in 1 : 1,000, all from Santa Cruz Biotech [Dallas, TX]), MMP-9 Ab (Abcam, Cambridge, UK), p65 Ab, and p53 Ab (Genetex, Irvine, CA). The primary antibodies were removed, and the membrane was washed extensively in PBST buffer. Subsequent incubation with horseradish peroxidase-conjugated goat anti-rabbit antibodies (1 : 20,000, Santa Cruz Biotech) was performed at room temperature for 2 hours. The membrane was washed extensively in PBST buffer to remove any excess secondary antibodies, and the blot was visualized with enhanced chemiluminescence reagent (GE Healthcare).
4
Western Blot Analysis of TM4SF1 Protein
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Total protein was isolated from cells or tissues using cell lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, and 1% Protease Inhibitor Cocktail). Lysates were analyzed by SDS-PAGE and transferred to PVDF membranes (0.45 μM, Millipore, Billerica, MA, USA). Membranes were blocked with 5% skimmed milk and then immunoblotted at 4°C using the following primary antibodies: anti-TM4SF1 (1:1,000, Abcam, Cambridge, MA, USA), anti-β-actin (1:500, Abcam). Membranes were probed at room temperature for 1 hr with horseradish peroxidase-conjugated goat anti-rabbit antibody (Santa Cruz, CA, USA). Blots were detected using the Dyne ECL STAR Western Blot Detection kit (Millipore). All experiments were performed in triplicate.
5
Quantification of HC1 and HC2 Proteins
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To quantify HC1 and HC2 protein expression, first instar larvae dissected from the uteri were homogenized twice in ice-cold lysis buffer which contained 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.5% Na-deoxycholate, 0.5% NP-40, 0.5% SDS, and protease inhibitor (EDTA-free). Lysates were cleared by centrifugation at 15.000×g at 4 °C for 30 min, and the protein content was determined by a protein assay using bovine serum albumin (BSA) as a standard (Bradford method). Sixty micrograms of protein from each sample was reconstituted directly in the appropriate amount of sample buffer and separated in Mini-Protean TGX System Precast Protein Gels (Bio-Rad, Hercules, CA, USA) and transferred to Trans-Blot Turbo Mini PVDF Transfer Packs (Bio-Rad, Hercules, CA, USA). The membranes were washed and blocked in 0.02 M Tris-buffered saline containing 5% BSA and 0.1% Tween 20 and then incubated overnight at 4 °C with anti-HC1 or HC2 antibodies diluted 1:500. Next, the membranes were washed with TBST (Tris-buffered saline containing 0.1% Tween 20) and incubated for 1 h with a horseradish peroxidase-conjugated goat anti-rabbit antibody (Santa Cruz, USA) diluted 1:1000. Signals were detected by chemiluminescence using WesternBright Quantum HRP substrate (Advansta Inc., Menlo Park, USA) and visualized using the Chemidoc™ XRS + System (Bio-Rad, Hercules, USA).
6
Western Blot Analysis of Lung Proteins
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Western blot analysis was performed using the NOVEX NuPAGE electrophoresis system (Invitrogen, Grand Island, NY) with 1.5 mm 4–12% Bis–Tris gels according to manufacturer's instructions. Briefly, 25 mg of lung homogeneous was loaded to each well and gels were run at 100 V at 4 °C for 2 h in NuPAGE MOPS SDS running buffer under reducing conditions. Proteins were transferred to nitrocellulose membrane at 80 V for 60 min at 4 °C. The membrane was then blocked for 1h at room temperature with 5% BSA in Tween/Tris buffered saline (TTBS; 100 mM Tris base, 1.5 M NaCl adjusted to pH 7.4 with 0.1% Tween 20). Immunoblots were then incubated with myc-tag antibody overnight at 4 °C. The following day, the membrane was washed with TTBS three times, for 20 min each time. A horseradish peroxidase-conjugated goat anti-rabbit antibody (1:10,000, Santa Cruz Biotechnology, Santa Cruz, CA), used as the secondary antibody, was applied for 1 h at room temperature. Following this, the membrane was washed with TTBS followed by two 10-min washes with TBS. The blots were developed using a chemiluminescence system (Amersham Pharmacia Biotech, Piscataway, NJ).
7
Protein Expression Analysis in Cells
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Total protein was isolated from cells using cell extraction buffer (Biosource, Camarillo, CA) supplemented with protease and phosphatase inhibitors. Protein concentrations were measured using a BCA Protein Assay kit (Pierce, Rockford, IL). Antibodies against IL13Rα2, TAZ, PI3K, Akt, CTGF, CD24, NEDD9 and β-actin were used as primary antibodies. The blots were incubated with a horseradish peroxidase-conjugated goat anti-rabbit antibody (Santa Cruz Biotechnology). Proteins were visualized with electrochemical luminescence (GE Healthcare, CA).
8
Protein Expression Analysis in Transfected Cells
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Transfected cells were lysed with RIPA buffer at 72 hours after transfection, and the protein concentration was determined using a Pierce BCA protein assay reagent kit (Pierce). Proteins (30 µg) were separated by electrophoresis on 8% to 10% sodium dodecyl sulfate polyacrylamide gels and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% milk in tris-buffered saline with Tween 20 (TBST) at room temperature for 30 minutes and incubated with the appropriate primary antibody as follows: Bax (Santa Cruz Biotechnology, Santa Cruz, CA, USA), PI3K (Santa Cruz Biotechnology), p-Akt (Santa Cruz Biotechnology), p-GSK3β (Santa Cruz Biotechnology), or GAPDH (Santa Cruz Biotechnology) at 4°C overnight. The membranes were washed with TBST and incubated with a horseradish peroxidase-conjugated goat anti-rabbit antibody (Santa Cruz Biotechnology) for 1 hour at room temperature. The protein blank was detected using enhanced chemiluminescence (ECL Plus™; GE Healthcare, Little Chalfont, UK).
9
Western Blot Analysis of BRD4 Expression
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Equal amounts of proteins (30 µg) from the lysates of the cells were subjected to electrophoresis through a 10% SDS-PAGE (Beyotime Institute of Biotechnology) at 80 V for 30 min and at 100 V for 1.5 h. The proteins were then transferred onto polyvinylidene difluoride membranes. After blocking in 5% skimmed milk, the membranes were then incubated with the following diluted primary antibodies: Rabbit polyclonal BRD4 (Abcam, Cambridge, MA, USA), mouse monoclonal β-actin (Beyotime Institute of Biotechnology) overnight at 4°C and horseradish peroxidase-conjugated goat anti-rabbit antibody (Santa Cruz Biotechnology, Inc.) at room temperature for 2 h. Specific bands were visualised on an autoradiographic film using an enhanced chemiluminescence reagent (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China).
10
Western Blot Analysis of Plk1 Expression
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Cells were washed three times with phosphate buffered saline (PBS, 4 °C), and cellular protein was subsequently collected by lysis in 80 µL of RIPA buffer at 4 °C for 20 minutes. Removal of cell debris was performed by centrifugation at 12,000×g and 4 °C for 15 minutes, and the supernatant was collected for subsequent analyses. Afterwards, the Bio-Rad protein assay was used to determine the protein concentrations, and the same amount of protein (40 µg) per group was separated by electrophoresis, and subsequently transferred to polyvinylidene fluoride membranes (Amersham, USA). The membranes were blocked by incubation in 5% non-fat milk in Trisbuffered saline for 1 hour, followed by incubation with primary antibodies overnight at 4 °C. Anti-Plk1 monoclonal antibody (Cell Signaling Technology, Inc., USA) was used at 1 µg/mL. The membranes were then incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit antibody (Santa Cruz Biotechnology, Inc., USA) at a 1:30,000 dilution. Electrochemiluminescence was detected on a ChemiDoc XRS+ Gel Imaging System. Next, in order to ensure that the same amount of protein had been loaded, the membranes were washed three times with Trisbuffered saline and re-incubated with an antibody directed against GAPDH.
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