Hrp conjugated anti rabbit secondary antibody sc 2004

Manufactured by Santa Cruz Biotechnology

Sourced in Spain

The HRP-conjugated anti-rabbit secondary antibody (sc-2004) is a detection reagent used in various immunological techniques. It consists of a rabbit-specific secondary antibody conjugated to the enzyme horseradish peroxidase (HRP). This antibody can be used to detect and visualize primary antibodies raised in rabbits.

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Lab products found in correlation

Protease inhibitor, by Roche (2 mentions) Bca method, by Merck Group (2 mentions) A6397, by Thermo Fisher Scientific (2 mentions) Pi34095, by Thermo Fisher Scientific (2 mentions) Ecl reagent, by GE Healthcare (1 mentions) Ripa lysis buffer, by Roche (1 mentions) Anti mouse hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions) Pvdf membrane, by Cytiva (1 mentions) Enhanced chemiluminescent reagent, by GE Healthcare (1 mentions)

Spelling variants of this product

HRP-conjugated secondary antibodies (674 citations) Sc-2004 (82 citations) Anti-rabbit secondary antibody (54 citations) HRP-conjugated anti-rabbit secondary antibody (34 citations) HRP-conjugated anti-rabbit antibody (23 citations) Anti-rabbit (sc-2004) (14 citations) Goat anti-rabbit HRP-conjugated secondary antibody (sc-2004; (3 citations) HRP-conjugated secondary antibody (sc-2004) (2 citations) Rabbit anti-TM (2 citations)

4 protocols using hrp conjugated anti rabbit secondary antibody sc 2004

Protein Extraction and Quantification from Muscle Tissue

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For protein extraction, muscle powdered tissue was resuspended in RIPA lysis buffer containing protease inhibitors (Roche, Basel, Switzerland), homogenated and centrifuged at 10,000× g for 10 min at 4 °C. The supernatant was collected and the protein concentration was determined by BCA method (Sigma-Aldrich, San Luis, MO, USA). After quantification, 25 μg of protein were loaded to an 8–10% SDS-PAGE and transferred to PVDF membranes (Amersham™, GE Healthcare Life Sciences, Little Chalfont, UK). For immunodetection, membranes were blocked with a Tris-buffered saline solution containing 5% skimmed milk and 0.1% Tween for 1 h at room temperature and then incubated overnight at 4 °C with the selected primary antibodies (Table 3). GAPDH was selected as the normalization protein. The next day, membranes were washed and incubated with HRP-conjugated anti-rabbit secondary antibody (sc-2004; Santa Cruz Biotechnology, Quimigen S.L., Madrid, Spain) for 1 h at room temperature, and the protein bands were visualized using ECL reagents (GE Healthcare Life Sciences, Little Chalfont, United Kingdom). Densitometry study was performed using AlphaEase FC software (Bonsai Technologies Group, Madrid, Spain).

Moreno-García L., Miana-Mena F.J., Moreno-Martínez L., de la Torre M., Lunetta C., Tarlarini C., Zaragoza P., Calvo A.C, & Osta R. (2021). Inflammasome in ALS Skeletal Muscle: NLRP3 as a Potential Biomarker. International Journal of Molecular Sciences, 22(5), 2523.

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Microtubule Assembly Dynamics under Oxidative Stress

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A mixture of 36.5 μM unlabeled tubulin, 1mM GTP, and 50μM DCP-Rho1 in Brb80 was prepared on ice and incubated for 2.5h at 37°C in the presence or absence of H₂O₂ (0–1.5mM). The mixture was diluted 30-fold with warm 10μM Taxol in Brb80. 240μL of the microtubule sample were spun via air-driven centrifuge (20psi, 5min), and the pellet was re-suspended in 100μL of warm 10μM Taxol in Brb80 and stored at −80°C. Reducing electrophoresis buffer was added and heated to denature proteins, and immunoblotting was performed. Samples were resolved in 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked in 1% BSA in Tris-buffered saline with 0.15% Tween. For the detection of DCP-Rho1, lysates were probed with primary antibody against rhodamine (A6397, Fisher Scientific) followed by HRP-conjugated anti-rabbit secondary antibody (sc-2004, Santa Cruz Biotechnology). For detection of tubulin, membranes were probed with primary antibody against alpha-tubulin (clone DM1A, #PI34095, VWR) followed by HRP-conjugated anti-mouse secondary antibody (SC-205, Santa Cruz BioTechnology). Western blots were developed using chemiluminescence (PI34095, Fisher) and densitometry analysis was performed using ImageJ software.

Goldblum R.R., McClellan M., White K., Gonzalez S.J., Thompson B.R., Vang H.X., Cohen H., Higgins L., Markowski T.W., Yang T.Y., Metzger J.M, & Gardner M.K. (2021). Oxidative stress pathogenically remodels the cardiac myocyte cytoskeleton via structural alterations to the microtubule lattice. Developmental cell, 56(15), 2252-2266.e6.

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Detecting Oxidative Stress in H9c2 Cells

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H9c2 cells were grown in DMEM+ media for 3d, then passaged into 6-well dishes. Cells were incubated in 0–1mM H₂O₂ for 1h. During the final 10 min of the incubation in H₂O₂, 10 μM DCP-Rho1 in DMSO was added. Cells were released and lysed in hot, reducing electrophoresis buffer for 6min. Samples were resolved in 10% SDS-PAGE and transferred to PVDF membranes. Membranes were then blocked in 1% BSA in Tris-buffered saline with 0.15% Tween. For detection of DCP-Rho1, lysates were probed with primary antibody against rhodamine (A6397, Fisher Scientific) followed by HRP-conjugated anti-rabbit secondary antibody (sc-2004, Santa Cruz Biotechnology). For detection of tubulin, membranes were probed with primary antibody against alpha-tubulin (clone DM1A, #PI34095, VWR) followed by HRP-conjugated anti-mouse secondary antibody (SC-205, Santa Cruz BioTechnology). Western blots were developed using chemiluminescence (PI34095, Fisher) and densitometry analysis was performed using ImageJ software.

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Western Blot Analysis of hSOD1 Protein

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Powdered tissue (see above) was homogenized in RIPA lysis buffer containing protease inhibitors (Roche). The homogenate was centrifuged at 10000 ×g for 10 min at 4°C, the supernatant was collected and the protein concentration was determined by BCA method (Sigma Aldrich). Forty micrograms of total protein were subjected to SDS/PAGE and transferred to PVDF membranes (Amersham Biosciences). For immunodetection, membranes were blocked overnight in 5% skimmed milk at 4°C and then incubated one hour with the primary antibody anti-hSOD1 (HPA001401 Sigma-Aldrich). After washes, the membranes were incubated with HRP-conjugated anti-rabbit secondary antibody (sc-2004 Santa Cruz 1:3000), washed again and finally incubated with enhanced chemiluminescent reagent (GE Healthcare Life Science). Immunoblots were exposed and scanned, and quantitative densitometry was performed with AlphaEaseFC software (Bonsai).

Rando A., de la Torre M., Martinez-Muriana A., Zaragoza P., Musaro A., Hernández S., Navarro X., Toivonen J.M, & Osta R. (2019). Chemotherapeutic agent 5-fluorouracil increases survival of SOD1 mouse model of ALS. PLoS ONE, 14(1), e0210752.

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