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Anti rabbit hrp secondary antibody

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The Anti-rabbit HRP secondary antibody is a laboratory tool used in immunodetection techniques. It binds to rabbit primary antibodies and is conjugated with the enzyme horseradish peroxidase (HRP). This secondary antibody enables the detection and visualization of target proteins in various immunoassays, such as Western blotting, ELISA, and immunohistochemistry.

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Lab products found in correlation

β actin, by Cell Signaling Technology (3 mentions) Halt protease phosphatase inhibitor, by Thermo Fisher Scientific (2 mentions) Caspase 3, by Cell Signaling Technology (2 mentions) Ripa buffer, by Thermo Fisher Scientific (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Gw9662, by Merck Group (1 mentions) Cell culture reagents, by Thermo Fisher Scientific (1 mentions) Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions) P akt akt, by Cell Signaling Technology (1 mentions) West dura chemiluminescence substrate, by Thermo Fisher Scientific (1 mentions) Mtor p mtor, by Cell Signaling Technology (1 mentions) Stat3, by Cell Signaling Technology (1 mentions) Ab186838, by Abcam (1 mentions) Ab63576, by Abcam (1 mentions) Gapdh, by Cell Signaling Technology (1 mentions) Rhodamine 123, by Merck Group (1 mentions) Fetal bovine serum (fbs), by Merck Group (1 mentions) 2 7 dichlorodihydrofluorescein diacetate dcfh da, by Merck Group (1 mentions) P nf κb, by Cell Signaling Technology (1 mentions) Cycletest plus dna kit, by BD (1 mentions)

10 protocols using anti rabbit hrp secondary antibody

1

Investigating Anti-diabetic Potential of Antrodia camphorata

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The ethanolic extract of A. camphorata was provided by Biotech Lantyng Company (Taipei, Taiwan). Cell culture reagents were obtained from Gibco (USA). GW9662, Rosaglitazone, and MTT were purchased from Sigma (USA). PPAR-γ (D69), ki-67 (D3B5) mAb, Caspase-3, β-actin, α,β-Tubulin and GAPDH antibodies, and anti-rabbit-HRP secondary antibodies were obtained from Cell Signaling Technology (USA), while GLK, p-IRE1α (phospho-S724), IRE1α antibodies were obtained from Abcam (USA). p-PERK (phosphor-Thr981), PERK (aa947-996) antibodies were purchased from LifeSpan BioSciences (USA), while GLUT-2 (H-67) and ATF6α (H-280) antibodies were purchased from Santa Cruz Biotechnology (USA).
Vong C.T., Tseng H.H., Kwan Y.W., Lee S.M, & Hoi M.P. (2016). Antrodia camphorata Increases Insulin Secretion and Protects from Apoptosis in MIN6 Cells. Frontiers in Pharmacology, 7, 67.
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2

Profiling Protein Signaling in Keratinocytes

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Total protein lysates were prepared from pooled untreated or treated keratinocytes using RIPA buffer containing Halt protease/phosphatase inhibitors in accordance with the manufacturer’s protocol (Thermo Fisher Scientific, Rockford, IL, USA). 25 μg of protein was electrophoresed using 4–12% gradient SDS-polyacrylamide gels, transferred to PVDF membrane and blocked with 5% BSA. Primary antibodies for pEGFR/EGFR, MET, pHER2/HER2, pHER3/HER3, pAKT/AKT, STAT3, pmTOR/mTOR, B-Actin were purchased from Cell Signaling and used at 1:1000. p-MET was purchased from Abcam and used at 1:1000. Anti-rabbit HRP secondary antibodies (Cell Signaling Technology), followed by West Dura Chemiluminescence substrate (Thermo, Rockland, IL) were used for signal detection. Bands were quantified using NIH Image J and normalized to the densitometry for the respective housekeeping gene. Western blots used pooled keratinocyte protein (n = 4–6 mice/pool) and were repeated a minimum of three times.
Kelley M.B., Geddes T.J., Ochiai M., Lampl N.M., Kothmann W.W., Fierstein S.R., Kent V, & DeCicco-Skinner K. (2022). Loss of Tpl2 activates compensatory signaling and resistance to EGFR/MET dual inhibition in v-RAS transduced keratinocytes. PLoS ONE, 17(3), e0266017.
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3

Comprehensive Protein Expression Analysis of Skin Cell Types

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Total protein lysates were prepared from keratinocytes and fibroblasts using RIPA buffer containing Halt protease/phosphatase inhibitors in accordance with the manufacturer’s protocol (Thermo Fisher Scientific, Rockford, IL, USA). Twenty-five micrograms of protein was electrophoresed using 4–12% gradient SDS-polyacrylamide gels, transferred to PVDF membrane and blocked with 5% BSA. Primary antibodies for HGF (Abcam; cat #ab83760), TGFβ (Abcam; cat #ab92486), TGFβR1 (Abcam; ab31013), TGFβR2 (Abcam; ab186838), SMAD2 (Abcam; ab63576), SMAD4 (Abcam; ab236321) MET (Cell Signaling; cat #3127), p-MET (Abcam; ab5662), GAPDH (Cell Signaling, Cat #5174), B-actin (Cell Signaling; Cat #4970), tubulin (Cell Signaling, Cat #15115) were incubated with membranes at 1:1000. Anti-rabbit HRP secondary antibodies (Cell Signaling Technology), followed by West Dura Chemilluminescent substrate (Thermo, Rockland, IL) were used for signal detection. Bands were quantified using NIH Image J and normalized to the densitometry for the respective housekeeping gene. All Western blots used pooled protein from triplicate samples and were repeated a minimum of three times. Quantification of Western blots is displayed in Supplementary data A.
Bonan N.F., Kowalski D., Kudlac K., Flaherty K., Gwilliam J.C., Falkenberg L.G., Maradiaga E, & DeCicco-Skinner K.L. (2019). Inhibition of HGF/MET signaling decreases overall tumor burden and blocks malignant conversion in Tpl2-related skin cancer. Oncogenesis, 8(1), 1.
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4

Cytotoxicity Evaluation of Compounds

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Dulbecco’s modified Eagle’s medium (DMEM), Hoechst 33,342, Fluoromount, 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA), and Rhodamine-123 and Fetal Bovine Serum (FBS) were purchased from Sigma (St. Louis, MO, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and trypsin were procured from Hi-media Pvt. Limited, Mumbai (India). Rabbit monoclonal Bcl-xl, p-53, p-NF-κB, Caspase3, and Caspase9 antibodies and anti-rabbit- HRP secondary antibody were obtained from Cell Signaling Technology, Danvers, MA, USA. PVDF membrane (MDI, Ambala). The RT-PCR chemical kit was purchased from Bio-Rad, CA, USA. The BD Cycletest plus DNA Kit was from BD Biosciences, San Jose, CA, USA. Chemicals and reagents of analytical (AR) grade were used to perform the experiments.
Kumar A., Kaur S., Dhiman S., Singh P.P., Bhatia G., Thakur S., Tuli H.S., Sharma U., Kumar S., Almutary A.G., Alnuqaydan A.M., Hussain A., Haque S., Dhama K, & Kaur S. (2022). Targeting Akt/NF-κB/p53 Pathway and Apoptosis Inducing Potential of 1,2-Benzenedicarboxylic Acid, Bis (2-Methyl Propyl) Ester Isolated from Onosma bracteata Wall. against Human Osteosarcoma (MG-63) Cells. Molecules, 27(11), 3478.
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5

Quantifying Insoluble Amyloid-Beta Levels

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In order to determine the effect of IgG1-iS18 treatment on the insoluble Aβ levels, a dot blot was performed. The GuHCl protein extracts were dotted onto a nitrocellulose membrane and allowed to dry. The membrane was blocked in 3% BSA in PBST for 30 min where after it was incubated with rabbit anti-human Aβ 1-42 (Cell Signaling Technology®, D9A3A) (1:1000) at room temperature for 1 hour. The membrane was washed 3 x with PBST for 5 min and incubated with anti-rabbit HRP secondary antibody (Cell Signalling) (1:2500) at room temperature for 30 min. After the membrane was washed another 3 × with PBST, it was incubated with Clarity™ Western ECL Blotting Substrate (Biorad) and the ChemiDoc™ Imaging System (Biorad) was used for detection. Densitometric analysis was performed with Image Lab 5.1 software (Biorad).
Ferreira E., Bignoux M.J., Otgaar T.C., Tagliatti N., Jovanovic K., Letsolo B.T, & Weiss S.F. (2018). LRP/LR specific antibody IgG1-iS18 impedes neurodegeneration in Alzheimer's disease mice. Oncotarget, 9(43), 27059-27073.
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6

Protein Expression Analysis by Western Blot

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Protein was isolated using RIPA buffer and ran in a 17-well Bolt™ 4–12% Bis-Tris Plus Gels containing 1X Bolt™ MOPS SDS Running Buffer. Precision Plus Protein™ Dual Color Standard was used as a protein ladder and gels run for 32 min at 200 V.
Gels were transferred using an iBlot™ 2 PVDF Transfer Stack in an iBlot 2 Dry Blotting System. Antibodies were 1:200 rabbit anti-CACNA1C (Abcam, ab58552), 1:10 000 rabbit anti-vinculin (Abcam, ab129002), 1:500 rabbit anti-RGS4 (Abcam, ab9964), 1:X rabbit anti-CACNB1 (Sigma, AV34953), 1:X rabbit anti-GNB1 (ThermoFisher Scientific, PA1-725), and 1:500 rabbit anti-GNG12 (ThermoFisher Scientific, PA5-75620). Anti-rabbit HRP secondary antibody (1:2000, Cell Signalling Technologies, 7074S) was used. Membranes were developed using Clarity™ Western ECL Blotting Substrates and imaged using a ChemiDoc imaging system. Images were analysed using ImageJ with vinculin as the loading control.
Couch L.S., Fiedler J., Chick G., Clayton R., Dries E., Wienecke L.M., Fu L., Fourre J., Pandey P., Derda A.A., Wang B.X., Jabbour R., Shanmuganathan M., Wright P., Lyon A.R., Terracciano C.M., Thum T, & Harding S.E. (2021). Circulating microRNAs predispose to takotsubo syndrome following high-dose adrenaline exposure. Cardiovascular Research, 118(7), 1758-1770.
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7

Western Blot Analysis of Bscl2 in Frozen BAT Tissues

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Frozen BAT tissues were homogenised in RIPA lysis buffer containing cOmplete™ protease inhibitor cocktail (Roche). Protein concentrations were determined by BCA assay (Thermo). SDS-PAGE was performed using equal quantities of protein which were transferred to PVDF membrane using standard protocols. Antibodies used included anti-Bscl2 (ab106793, Abcam) and anti-Calnexin (ab75801, Abcam). Anti-Rabbit HRP secondary antibody was used (Cell Signaling) and was visualized using enhanced chemiluminescence (ECL substrate, Illuminata) and quantified by densitometry scanning using Image J software.
Mcilroy G.D., Suchacki K., Roelofs A.J., Yang W., Fu Y., Bai B., Wallace R.J., De Bari C., Cawthorn W.P., Han W., Delibegović M, & Rochford J.J. (2018). Adipose specific disruption of seipin causes early-onset generalised lipodystrophy and altered fuel utilisation without severe metabolic disease. Molecular Metabolism, 10, 55-65.
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8

Western Blot Analysis of Signaling Proteins

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Cells were treated with various concentrations of each tested compound for a designated time. Then, the cells were lysed using 1× SDS sample lysis buffer containing protease and phosphatase inhibitors. Cell lysates were loaded and electrophoresed onto 8–12% SDS-PAGE gels, and then the separated proteins were transferred to PVDF membranes, which were blocked with 5% fat-free milk in TBS solution containing 0.5% Tween-20 for 4 h at room temperature. Then, the membranes were incubated with the corresponding primary antibody (1:1000–1:200) overnight at 4 °C, after which they were washed with TBST and incubated with HRP-conjugated secondary antibody for 2 h. The protein signals were visualized by an ECL Western blotting detection kit (Thermo Scientific, Waltham. MA, USA) and detected with an Amersham Imager 600 system (GE, Boston, MA, USA). Commercial antibodies against PLCG1, beta-actin-HRP and anti-rabbit HRP secondary antibody were obtained from Cell Signaling Technology. The CDK1 and Caspase3 antibody were obtained from Abcam Technology.
Li T., Yang Z., Li H., Zhu J., Wang Y., Tang Q, & Shi Z. (2021). Phospholipase Cγ1 (PLCG1) overexpression is associated with tumor growth and poor survival in IDH wild-type lower-grade gliomas in adult patients. Laboratory Investigation; a Journal of Technical Methods and Pathology, 102(2), 143-153.
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9

Quantifying EV CD9 Expression

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3.108 total particles were loaded into pre-cast gels (BioRad, Cat. # 4561094). Samples were blocked in a 5% nonfat milk solution and stained for the common EV marker CD9 using a primary antibody (Cell Signaling Technologies, Cat. # 13174) and an anti-rabbit HRP secondary antibody (Cell Signaling Technologies, Cat. # 7074). Samples were imaged using the BioRad Chemidoc chemiluminescent system. FIJI was used to quantify the intensity of bands in Western blots to determine CD9 expression fold difference among EV samples.
Walker S.N., Lucas K., Dewey M.J., Badylak S., Hussey G., Flax J, & McGrath J.L. (2024). Rapid Assessment of Biomarkers on Single Extracellular Vesicles Using ‘Catch and Display’ on Ultrathin Nanoporous Silicon Nitride Membranes. bioRxiv.
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10

Western Blot Analysis of TLR2 Expression

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Cell pellets were collected from enteroids monolayers and solubilized in RIPA lysis buffer containing protease and phosphatases inhibitor (Roche), followed by homogenization by sonication. After centrifugation at 5000 g for 10 min, the supernatant was collected as the protein lysate. Protein concentrations were determined using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Twenty-five micrograms of protein from each sample were resolved onto 4 to 15% SDS-PAGE gradient gels (Bio-Rad). Following electrophoresis, proteins were transferred to nitrocellulose membrane. After blocking with 5% non-fat milk in TBST, blots were probed with 1 : 500 dilution of primary antibody (TLR2, cat# MA5–32787, Invitrogen) overnight at 4°C, followed by a 1 : 2000 dilution of anti-rabbit HRP secondary antibody (Cat# 7074, Cell Signaling Technology) for 1 h at room temperature. SuperSignalTM West Femto Maximum Sensitivity Substrate (Thermo Fisher) was applied and the blot was imaged on a chemiluminescent imaging system. The blot was then stripped and probed with HRP-conjugated mouse anti-beta actin diluted 1 : 3000 (Cell Signaling Technology) before substrate addition and imaging. Beta actin was used as the loading control. Image J is used to analyze the band intensity.
Das T.K., Blasco-Conesa M.P., Korf J., Honarpisheh P., Chapman M.R, & Ganesh B.P. (2022). Bacterial Amyloid Curli Associated Gut Epithelial Neuroendocrine Activation Predominantly Observed in Alzheimer’s Disease Mice with Central Amyloid-β Pathology. Journal of Alzheimer's disease : JAD, 88(1), 191-205.
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