Primary antibodies used for western blot experiments: phospho-p53 Ser15, cleaved Caspase-3 and cleaved PARP (Cell Signaling, Leiden, The Netherlands), MAPK15 (custom preparation), p53 (Santa Cruz Biotechnology), ACTB (Sigma Aldrich), CDKN1A/p21 (Epitomics, Burlingame, CA), LC3B (Novus Biologicals, Cambridge, UK), SQSTM1/p62 (BD Biosciences, Milan, Italy), GADD45a (Cell Signaling), phospho-ATR (Cell Signaling), phospho-CHK2 (Cell Signaling), PCNA (Cell Signaling). Primary antibodies used for immunofluorescence experiments: γH2A.X (Cell Signaling), 53BP1 (Novus Biologicals) LC3B (MBL, Woburn, MA). Primary antibody used for immunohistochemistry experiments: MAPK15 (custom preparation). Secondary antibodies used for western blot experiments: HRP-conjugated anti-Mouse IgG and HRP-conjugated anti-Rabbit IgG (Santa Cruz Biotechnology). Secondary antibodies used for immunofluorescence experiments: AlexaFluor488-conjugated anti-Rabbit IgG and AlexaFluor555-conjugated anti-Mouse IgG (Life Technologies).
Hrp conjugated anti rabbit igg
Manufactured by Santa Cruz Biotechnology
Sourced in United States
HRP-conjugated anti-rabbit IgG is a secondary antibody conjugated with horseradish peroxidase (HRP). It is designed to detect and bind to rabbit primary antibodies in immunoassays and other immunological applications.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated anti mouse igg, by Santa Cruz Biotechnology (9 mentions)
Protein assay kit, by Bio-Rad (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Anti gapdh, by Santa Cruz Biotechnology (3 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Nf κb p65, by Santa Cruz Biotechnology (2 mentions)
Z vad fmk, by R&D Systems (2 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
Vimentin, by Cell Signaling Technology (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Chemidoc xrs system, by Bio-Rad (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Ecl western blotting kit, by Cytiva (2 mentions)
Polyvinylidene difluoride membrane, by Pall Corporation (2 mentions)
Sqstm1 p62, by BD (1 mentions)
Gadd45a, by Cell Signaling Technology (1 mentions)
Phospho atr, by Cell Signaling Technology (1 mentions)
Phospho chk2, by Cell Signaling Technology (1 mentions)
37 protocols using hrp conjugated anti rabbit igg
1
Antibody Characterization for Cell Signaling
2
Western Blot Analysis of FAK Signaling
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Total cell protein extracts were prepared using RIPA buffer as described previously [12 (link)]. Membranes were blocked for 1 h with 10% non-fat milk or 5% BSA in TBS containing 0.1% Tween-20, and then incubated overnight at 4°C with the primary antibody at the dilutions recommended by the manufacturer.
The primary antibodies against FAK and phospho-FAK (Tyr397) were purchased from Cell Signaling (Danvers, MA, USA), and the anti-β-actin from Sigma-Aldrich (Steinheim, Germany). HRP-conjugated anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as the secondary antibody. Blots were developed using Lumi-Light Plus Reagent (Roche, Barcelona, Spain), and the autoradiograms were scanned using a GS-800 calibrated densitometer and analyzed using Quantity One software (Biorad, Berkeley, CA,USA).
The primary antibodies against FAK and phospho-FAK (Tyr397) were purchased from Cell Signaling (Danvers, MA, USA), and the anti-β-actin from Sigma-Aldrich (Steinheim, Germany). HRP-conjugated anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as the secondary antibody. Blots were developed using Lumi-Light Plus Reagent (Roche, Barcelona, Spain), and the autoradiograms were scanned using a GS-800 calibrated densitometer and analyzed using Quantity One software (Biorad, Berkeley, CA,USA).
3
Western Blotting Technique and Analysis
4
Protein Expression Analysis in Cardiac Tissue
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After lysis buffer was added to cardiac tissue, total protein was extracted, and the concentration was measured. Equal amounts of protein preparations were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 30 min at 80 V; then, the separated proteins were transferred to nitrocellulose membranes (Boer Biotechnology Company) for 60 min at 120 V. The membranes were blocked with 5% nonfat milk (SIGMA, USA) in PBS with 0.05% Tween-20 (PBST, pH 7.6) for 2 h and then incubated with the following primary antibodies: APPL1 (1 : 500, Santa Cruz), p-AMPK and T-AMPK (1 : 1000, Santa Cruz, USA), PPARα (1 : 500 and 1 : 1000, Santa Cruz, USA), and NF-κB (P65) (1 : 600, Santa Cruz, USA) at 4°C overnight. The membranes were washed with TBST and then incubated with 1 : 5000 HRP-conjugated antirabbit IgG (Santa Cruz, USA) for 90 min on a tabletop incubator at 50 rpm and 37°C. The membranes were then washed again with TBST. The membranes were scanned with Typhoon (Pharmacia, USA) and quantitated using Quality One. We detected the protein expression level 3 times for each sample.
5
Western Blot Analysis of UCP1 Protein
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The protein concentration was determined by using a protein assay kit (Bio-Rad laboratories, Hercules, CA, USA). Proteins were diluted with Laemmli SDS-PAGE sample buffer and bolied for 5 min in the presence of 2-mercaptoethanol. The samples underwent sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 12.5% gel, followed by transfer to an Amersham Hybond-LFP polyvinylidene fluoride (PVDF) membrane (GE Healthcare, Little Chalfont, UK). The membranes were blocked with 5% skimmed milk and 0.1% tween-20 in PBS for 1 h, as previously described61 (link). After blocking, the membranes were incubated in a rabbit anti-UCP1 antibody (Sigma) overnight at 4 °C. Next, the membranes were incubated in HRP-conjugated anti-rabbit IgG (Santa Cruz Biotechnology, San Antonio, TX, USA) for 2 h at room temperature. Protein bands were visualized by ELC chemiluminescence detection (Millipore, Bedford, MA, USA). Immunoreactive protein bands were quantified by using the Image J software (NIH, Bethesda, MD, USA).
6
Protein Immunoblotting and Immunostaining
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The following reagents were purchased from the indicated providers: dimethyl sulfoxide (DMSO; Sigma, D8418) and mifepristone (RU-486; Sigma, M8046). The following antibodies were used for immunoblotting: mouse anti-TurboGFP (Origene, TA150041), rabbit anti-TDP-43 (Proteintech, 10782-2-AP), rabbit anti-LC3 (MBL, PM036), mouse anti-Polyubiquitin (Enzo Life Science, BML-PW8805), mouse anti-Flag (Cell Signaling Technology, 2044), rabbit anti-HDAC6 (Santa Cruz Biotechnology, sc-11420), mouse anti-Lamin A/C (EMD Millipore, 05-714), HRP-conjugated anti-alpha-tubulin (Cell Signaling Technology, 9099), HRP-conjugated anti-rabbit IgG (Santa Cruz Biotechnology, sc-2004), HRP-conjugated mouse IgM (Abcam, ab97230), and HRP-conjugated mouse IgG (Santa Cruz Biotechnology, sc-2005). The following antibodies were used for immunocytochemistry (ICC): rabbit anti-cleaved caspase-3 (CC3) antibody (Cell Signaling Technology, 9664) and Alexa 594-conjugated anti-rabbit IgG (Jackson ImmunoResearch, 111-585-144). The following antibodies were used for immunohistochemistry: rat anti-ELAV (DSHB, RAT-ELAV-7), mouse anti-Polyubiquitin (Enzo Life Science, BML-PW8805), Alexa-488 conjugated rat IgG (Jackson ImmunoResearch, 112-545-167), and Alexa-594 conjugated mouse IgM (Jackson ImmunoResearch, 115-587-020).
7
Caspase-Mediated Apoptosis Pathway Modulation
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Chloroquine (CQ), N-acetyl-L-cysteine (NAC) and acridine orange hemi (Zinc chloride) salt were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Caspase-8,-9, or -3 colorimetric assay kit and Z-VAD-FMK (a pan-caspase inhibitor) were obtained from R&D systems (MA, USA). Cycletest Plus DNA kit and FITC-Annexin V were purchased from BD bioscience Pharmingen (San Jose, CA, USA). Wortmannin and U0126 were purchased TOCRIS (Bristol, UK). 2’7’-dichlorofluorescein diacetate, BAPTA-AM, Ru360 and JC-1 were obtained from Calbiochem (San Diego, CA, USA). Dihydroethidium, lipofectamine 2000, anti-Alexa Fluor 488, Fluo4-AM and BAPTA were purchased from Invitrogen (Carlsbad, CA, USA). Rhod2 and Ruthenium Red were obtained from Abcam (Cambridge, UK). The antibodies used in this study are as follows; anti-caspase-3, anti-caspase-9, anti-caspase-8, anti-PARP, anti-ATG5, anti-total ERK, anti-p-ERK (Cell signaling Technology, MA, USA), LC3B/MAP1LC3B (NOVUS Biologicals, USA), anti-p62 lck ligand (BD bioscience Pharmingen, CA, USA), HRP-conjugated anti-rabbit IgG and HRP-conjugated anti-mouse IgG (Santa Cruz Biotechnologies, CA, USA).
8
Western Blot Analysis of Frizzled-1 Signaling
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Total ventricular extracts were prepared from the LV myocardium as described previously [31 (link)]. Protein concentrations were measured by BCA (Sigma-Aldrich, USA) assay. Equal amount of protein extracts was separated with SDS-page and transferred to a nitrocellulose membrane (Millipore, USA). The following primary antibodies were used in the present study: anti-GAPDH (USBiological, USA), anti-FZD1 (R&D systems, USA), anti-active-β-catenin (Millipore, USA), anti-β-catenin (Santa Cruz Biotechnology, USA), anti-p-GSK-3β (Cell-Signaling Technology, USA), anti-GSK-3β (BD Biosciences, Germany). HRP-conjugated anti-rabbit IgG, anti-mouse IgG, and anti-goat IgG antibodies (Santa Cruz Biotechnology, USA) were used as secondary antibodies. The blots were analyzed and quantified by densitometry using Image J program.
9
Protein Expression Analysis of Cell Lines
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Cells from each cell line were harvested and lyzed, and the total protein extracted using RIPA buffer (Beyotime, Shanghai, China). The protein was then quantified using bicinchoninic acid Protein Assay kit (Beyotime, Shanghai, China). Denatured protein samples (20 μg per lane) were separated by SDS‐PAGE using a 10% gel, and transferred to PVDF membranes (EMD Millipore). The membranes were blocked with 5% skim milk for one hour at room temperature, and incubated with primary antibodies against ACTL8 (1:500; cat. no. ab96756; Abcam), N‐cadherin (1:1000; cat. no. 4061; Cell Signaling Technology, Inc.), E‐cadherin (1:1000; cat. no. 3195; Cell Signaling Technology, Inc.), vimentin (1:2000; cat. no. ab92547; Abcam), β‐catenin (1:1000; cat. no. 9562; Cell Signaling Technology, Inc.), apoptosis regulator BAX (1:2000; cat. no. ab53154; Abcam), caspase 3 (1:1000; cat. no. ab49822; Abcam) and GAPDH (1:2500; cat. no. sc‐32 233; Santa‐Cruz Biotechnology, Inc.). After washing three times in PBST (1 × PBS, 0.1% Tween 20), the membranes were further incubated with secondary antibodies (1:2000) for 1.5 hours at room temperature (HRP‐conjugated anti‐rabbit IgG, sc‐2004, Santa‐Cruz; HRP‐conjugated anti‐mouse IgG, sc‐2005, Santa‐Cruz). All blots were visualized using Pierce ECL Western Blotting Substrate kit (Thermo Fisher Scientific, CA, USA).
10
Western Blot Analysis of Metabolic Proteins
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Proteins extracted from cells and animal tissues were separated by SDS–Polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted on to PVDF membrane (Millipore, IPVH00010) following standard protocols. Primary antibodies used in this study were anti-Insulin (Santa Cruz Biotechnology USA; sc- 25840), anti-Glucagon (Santa Cruz biotechnology USA; sc-74825), anti-G6PASE-α (Santa Cruz Biotechnology USA; sc- 25840), anti-PEPCK-C (Santa Cruz biotechnology USA; sc-74825), anti-AKT (Santa Cruz Biotechnology USA; sc-8312), anti-CASPASE 3 (Pierce USA; PA5–16335) and anti-GAPDH (Santa Cruz Biotechnology USA; sc-25778) and secondary antibody used was HRP conjugated anti-rabbit IgG (Santa cruz Biotechnology USA; sc-2030). Chemiluminescence detection was performed with the chemiluminescence detection kit (AmershamTM Western Blotting Detection Reagents, UK) according to the manufacturer’s instructions. The protein bands in western blots were quantified using Image J software.
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