Hrp enhanced chemiluminescent substrate

Manufactured by Thermo Fisher Scientific

Sourced in United States

The HRP-enhanced chemiluminescent substrate is a laboratory reagent used for the detection and quantification of horseradish peroxidase (HRP) in various immunoassay and Western blotting applications. It provides a sensitive and reliable method for the visualization of HRP-labeled target proteins or analytes.

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Lab products found in correlation

Dynabeads m 280 streptavidin, by Thermo Fisher Scientific (2 mentions) Silverquest silver staining kit, by Thermo Fisher Scientific (2 mentions) Protease inhibitor cocktail, by Merck Group (2 mentions) Chemiluminescence imaging system, by Bio-Rad (2 mentions) Ultracentrifuge tube, by Beckman Coulter (1 mentions) Sw60ti rotor, by Beckman Coulter (1 mentions) Chemidoc mp imaging system, by Bio-Rad (1 mentions) Ab157592, by Abcam (1 mentions) Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions) Ripa buffer, by Solarbio (1 mentions) P nf κb p65, by Cell Signaling Technology (1 mentions) Pmsf protease inhibitor, by Roche (1 mentions) E cadherin, by Cell Signaling Technology (1 mentions) Appropriate secondary antibody, by Cell Signaling Technology (1 mentions) β actin, by Cell Signaling Technology (1 mentions)

Close spelling variants of this product

HRP enhanced SuperSignal West Pico Chemiluminescent Substrate Pierce Enhanced chemiluminescent HRP substrate Enhanced chemiluminescent substrate for detection of HRP Enhanced chemiluminescent substrate Chemiluminescent HRP substrate Chemiluminescent substrate HRP substrate

8 protocols using hrp enhanced chemiluminescent substrate

Annexin V Binding Assay Protocol

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Example 5

20 μL of the pooled fractions from the sucrose gradient was mixed with 20 μL of 5× Annexin V binding buffer (Calbiochem) and made up to a final volume of 100 μL with water. 90 μL of the pooled fraction sample was incubated with 10 μL of Annexin V for 1 hour at room temperature with shaking at 800 rpm.

After washing 100 μl of Dynabeads® M-280 Streptavidin (Invitrogen) three times with 100 μl PBS, the reaction mix was added and incubated with shaking at 800 rpm for 30 mins. The beads were immobilised with a magnet and the supernatant or unbound fraction was removed. The beads were then washed four times with 100 μl PBS and the washes were removed each time after immobilizing the beads with a magnet.

The beads were boiled in 100 μl of a denaturing/reducing SDS-PAGE loading buffer to elute the remaining bound proteins. Equal volume of the unbound fraction, washes, eluted and bound fraction was resolved on 4-12% SDS-polyacrylamide gels. The gels were electroblotted onto a nitrocellulose membrane.

The membrane was probed with 1:50 dilution of mouse anti-human CD9 antibody. The secondary antibody was 1:1250 of HRP conjugated donkey anti-mouse IgG antibody. All antibodies were purchased from Santa Cruz.

The bound antibodies were visualized using HRP-enhanced chemiluminescent substrate (Thermo Fisher Scientific Inc., Waltham, Mass.) and exposure to an X-ray film.

US09977032B2. Microparticle fractionation (2018-05-22). Agency for Science, Technology and Research [SG], Singapore Health Services Pte Ltd. [SG]. Inventors: Sai Kiang Lim [SG], Kok Hian Tan [SG].

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Cholera Toxin Subunit B Affinity Purification

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Example 3

200 μL of serum flow through from the size exclusion were incubated with 0.1 g biotinylated Cholera Toxin subunit B (CTB) (Invitrogen) in 100 μL PBS pH 7.4 for 1 hour at room temperature with shaking at 800 rpm. After washing 100 d of Dynabeads® M-280 Streptavidin (Invitrogen) three times with 100 al PBS, the reaction mix was added and incubated with shaking at 800 rpm for 30 mins.

The beads were immobilised with a magnet and the supernatant or unbound fraction was removed. The beads were then washed twice with 100 μl PBS and the washes were removed each time after immobilizing the beads with a magnet. The beads were boiled in 100 μl of a denaturing/reducing SDS-PAGE loading buffer to elute the remaining bound proteins. Equal volume of the unbound fraction, washes, eluted and bound fraction was resolved on 4-12% SDS-polyacrylamide gels.

The gels were either stained with silver or electroblotted onto a nitrocellulose membrane. The membrane was probed with 1:50 dilution of mouse anti-human CD9 antibody. The secondary antibody was 1:1250 of HRP conjugated donkey anti-mouse Ig G antibody. All antibodies were purchased from Santa Cruz. The bound antibodies were visualized using HRP-enhanced chemiluminescent substrate (Thermo Fisher Scientific Inc., Waltham, Mass.) and exposure to an X-ray film.

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Sucrose Gradient Fractionation of Human Serum Proteins

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Example 4

14 sucrose solutions with concentrations from 22.8% to 60% were prepared and layered sequentially in an ultracentrifuge tube (Beckman Coulter Inc., CA) starting with the most concentrated solution. 500 μL of the human serum was loaded on top before ultracentrifugation for 16.5 h at 200 000 g, 4° C. in a SW60Ti rotor (Beckman Coulter Inc.). After centrifugation, 320 μL of 13 fractions were collected starting from the top of the gradient.

The densities of each fractions were determined by weighing a fixed volume. 20 μL of each fractions was resolved on 4-12% SDS-polyacrylamide gels. The gels were either stained with SilverQuest™ Silver Staining Kit (Invitrogen, Carlsbad, Calif.) or electroblotted onto a nitrocellulose membrane. The membrane was probed with either a 1:50 dilution of mouse anti-human CD9 antibody followed by a 1:1250 dilution of a HRP-conjugated donkey anti-mouse IgG antibody. All antibodies were purchased from Santa Cruz.

The bound antibodies were visualized using HRP-enhanced chemiluminescent substrate (Thermo Fisher Scientific Inc., Waltham, Mass.) and exposure to an X-ray film. Fractions 9 to 13 from the sucrose gradient were pooled and dialysed against PBS pH7.4 overnight. The pooled fractions were concentrated to 20 μL

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Protein Extraction and Analysis from Serum

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Example 2

2 mL Sepharose® 2B resins (Sigma Aldrich, Cat no. 2B300) was pipetted into a spin column (Bio-Rad, Cat no. 732-6008) and spun d at 800 g for 1 minute. The supernatant was removed and washed by adding 1 mL Phosphate Buffer Saline (PBS) followed by a 1-minute spin at 800 g. 1 mL of human serum was then loaded and the column was spun at 800 g for 1 minute.

The flow through was collected and this process was repeated thrice using 1 mL of PBS each time. 3 wash fractions were collected. 20 μL of each fraction was resolved on a 4-12% SDS/polyacrylamide gels. The gels were either stained with SilverQuest™ Silver Staining Kit (Invitrogen, Carlsbad, Calif.) or electroblotted onto a nitrocellulose membrane. The membrane was probed with either a 1:50 dilution of mouse anti-human CD9 antibody followed by a 1:1250 dilution of a HRP-conjugated donkey anti-mouse IgG antibody.

All antibodies were purchased from Santa Cruz. The bound antibodies were visualized using HRP-enhanced chemiluminescent substrate (Thermo Fisher Scientific Inc., Waltham, Mass.) and exposure to an X-ray film.

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Smyd3 Protein Expression Analysis

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Cells were collected from the plates and washed 3 times with PBS, followed by lysis in 10 volumes of modified RIPA buffer containing 50 mM Tris (pH 7.5), 1% NP40, 0.25% Na-Deoxycholate, 150 mM NaCl, 1mM EDTA, 10% Glycerol, 1 mM NaF and protease inhibitors cocktail (Roche). After 20 min. incubation at 4 ^oC, extracted proteins were recovered by centrifugation at 14000 rpm. The extracts were resolved by 10% SDS-PAGE, transferred to Protran Nitrocellulose membrane. Following transfer, the membranes were washed with TBST (10 mM Tris (pH 8.0), 150 mM NaCl and 0.1% Tween 20) and blocked in TBST buffer containing 5% low-fat dry milk for 1 hour at room temperature. After washing with TBST, the membranes were stained with Smyd3 antibody (1:1000 dilution) in TBST-1% milk overnight at 4 ^oC. The membranes were washed with TBST and stained with the HRP-conjugated anti rabbit IgG secondary antibody for 1 hour at room temperature.
The filters were probed with Enhanced Chemiluminescent HRP substrate (ThermoFisher) and detected using ChemiDoc MP imaging system (BioRad).

Kontaki H., Koukaki M., Vasilarou M., Giakountis A., Deligianni E., Luo X., Kim Y, & Talianidis I. (2021). Targeting Smyd3 by next-generation antisense oligonucleotides suppresses liver tumor growth. iScience, 24(5), 102473.

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Western Blot Analysis of WASH Protein

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Cells were lysed with RIPA buffer (Solarbio, China) containing protease inhibitor cocktail (Sigma-Aldrich). Cell lysates were sonicated on ice, resolved on SDS-PAGE and transferred to a nitrocellulose membrane. After blocking with 5% non-fat milk, the membranes were probed with primary antibodies for the detection of WASH (ab157592, Abcam) and β-actin (3700S, Cell Signaling Technology) followed by horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology). Protein expression was detected using enhanced chemiluminescent HRP substrate (Thermo Fisher Scientific) and photographed with a Chemiluminescence Imaging System (Bio-Rad Laboratories).

Guo M., Lian J., Liu Y., Dong B., He Q., Zhao Q., Zhang H., Qi Y., Zhang Y, & Huang L. (2022). Loss of miR-637 promotes cancer cell stemness via WASH/IL-8 pathway and serves as a novel prognostic marker in esophageal squamous cell carcinoma. Biomarker Research, 10, 77.

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Immunoblotting for Protein Analysis

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Immunoblotting was performed according to a standard protocol. Briefly, the cells were lysed with a RIPA lysis buffer (150 mM NaCl, 0.5% deoxycholic acid, 50 mM Tris-HCl [pH 7.5], 1% NP-40, and 0.1% sodium dodecyl sulphate [SDS]) containing a protease inhibitor mixture comprising 1 mM Na₃VO₄, 10 mM NaF, and 1 mM PMSF protease inhibitor (Boehringer Mannheim, Indianapolis, IN, USA). Cell lysates that were boiled in sample buffer were run through SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride membranes. Nonspecific reactions of the membranes were blocked with 5% skim milk, followed by incubation with primary antibodies overnight at 4°C. Antibodies for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), NF-κB p65, p-NF-κB p65, and E-cadherin were purchased from Cell Signaling Technology (Danvers, MA, USA). The incubated membranes were reacted with secondary antibodies in 5% skim milk at room temperature for 1 hour. Horseradish peroxidase (HRP)-linked immunoglobulin G (IgG) for various animals was purchased from Cell Signaling Technology. The blots were developed using an enhanced chemiluminescent HRP substrate (Thermo-Fisher Scientific/Invitrogen, Waltham, MA, USA).

Oh C., Kim H.J, & Kim H.M. (2019). Vitamin D maintains E-cadherin intercellular junctions by downregulating MMP-9 production in human gingival keratinocytes treated by TNF-α. Journal of Periodontal & Implant Science, 49(5), 270-286.

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Western Blot Analysis of WASH Protein

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Cells were lysed in ice‐cold lysis buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris, 1% NP‐40, 0.5% sodium deoxycholate, 0.1% SDS) plus protease inhibitor cocktail (Sigma‐Aldrich, St Louis, MO, USA). An equal amount of total protein from each sample (50 μg) was resolved on 10% SDS‐PAGE gel and then transferred to nitrocellulose membrane. The membrane was blocked with 5% non‐fat milk and then incubated with commercially available primary antibodies for the detection of WASH (Abcam, Cambridge, UK) and β‐actin (Cell Signaling Technology, Beverly, MA, USA), followed by the appropriate secondary antibodies (Cell Signaling Technology). Protein expression was detected using enhanced chemiluminescent HRP substrate (Thermo Fisher Scientific) and photographed with a Chemiluminescence Imaging System (Bio‐Rad Laboratories, Hercules, CA, USA).

Huang L., Lian J., Chen X., Qin G., Zheng Y, & Zhang Y. (2017). WASH overexpression enhances cancer stem cell properties and correlates with poor prognosis of esophageal carcinoma. Cancer Science, 108(12), 2358-2365.

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