Recombinant condensin II holo(WT) at 500 nM was mixed with or without λ protein phosphatase (NEB) at a final concentration of 400 U/µL in 1 x NEBuffer for Protein MetalloPhosphatases supplemented with 1 mM MnCl2 and incubated at 30°C for 60 min. Aliquot samples were taken at 0 and 60 min. Samples were analyzed by immunoblotting. The protein samples were subjected to 7.5% SDS-PAGE and transferred onto a nitrocellulose membrane (Cytiva). The membrane was blocked with 5% skim milk in TBS-T (25 mM Tris [pH 7.5], 0.15 M NaCl, and 0.05% Tween 20) before probing with appropriate affinity-purified rabbit hCAP-H2 antibody in 1% BSA in TBS (25 mM Tris [pH 7.5] and 0.15 M NaCl) followed by horseradish peroxidase-conjugated anti-rabbit IgG (Vector Laboratories) in TBS-T. After washing, the membrane was imaged with Amersham Imager 680 (Cytiva).
Horseradish peroxidase conjugated anti rabbit igg
Manufactured by Vector Laboratories
Sourced in United Kingdom
Horseradish peroxidase-conjugated anti-rabbit IgG is a secondary antibody conjugated with the enzyme horseradish peroxidase. It is used to detect and quantify the presence of rabbit immunoglobulin G (IgG) in various laboratory techniques, such as Western blotting, enzyme-linked immunosorbent assays (ELISA), and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Cytiva (3 mentions)
Horseradish peroxidase conjugated anti mouse igg, by Vector Laboratories (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Amersham imager 680, by Cytiva (1 mentions)
Enhanced chemiluminescence substrate, by Thermo Fisher Scientific (1 mentions)
Cell extraction buffer, by Thermo Fisher Scientific (1 mentions)
Detergent compatible protein assay, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Irdye 680rd goat anti rabbit igg, by LI COR (1 mentions)
Anti pstair, by Abcam (1 mentions)
Irdye 800cw goat anti mouse igg, by LI COR (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Complete mini edta free, by Roche (1 mentions)
Horseradish peroxidase conjugated anti mouse igg, by Merck Group (1 mentions)
Immobilon chemiluminescent horseradish peroxidase substrate, by Merck Group (1 mentions)
Bradford reagent, by Merck Group (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Ecl plus, by GE Healthcare (1 mentions)
Las 4000, by Fujifilm (1 mentions)
7 protocols using horseradish peroxidase conjugated anti rabbit igg
1
Dephosphorylation of Recombinant Condensin II
2
Quantifying IL-8 Protein Expression
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Total protein was extracted using a cell extraction buffer (BioSource International, Camarillo, CA) containing 3 mM phenylmethylsulfonyl fluoride and a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). The protein level was determined using a detergent-compatible protein assay (Bio-Rad, Hercules, CA). Samples (40 µg) were loaded on 10% Tris-hydrochloric acid-ready gels (Bio-Rad), electrophoretically separated, and electroblotted onto a nitrocellulose membrane (Bio-Rad). The membrane was blocked with 5% non-fat milk powder in TBS containing 0.1% Tween 20 (TBS-T) for 1 h to reduce any non-specific antibody binding. Subsequently, the membrane was incubated overnight with a monoclonal mouse IgG1 clone primary antibody against IL-8 (1:800 R&D Systems, Inc., Minneapolis, MN) in 5% non-fat milk powder in TBS-T. The membrane was then rinsed several times with 1x TBS-T for 1 h and incubated with horseradish peroxidase-conjugated anti-rabbit IgG (Vector Labs) in TBS-T. Following several washes, IL-8 was visualized through light emission from the film (Denville Scientific, Holliston, MA) with enhanced chemiluminescence substrate (Thermo Scientific, Rockford, IL). Band intensities were quantified using computer densitometry analysis (Image J, National Institutes of Health, Bethesda, MD).
3
Comprehensive Antibody Characterization Protocol
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Primary antibodies used in the current study were as follows: anti-XCAP-C (in-house identifier: AfR8) [33 (link)]; anti-CAP-E (in-house identifier: AfR9) [33 (link)]; anti-XCAP-D2 (in-house identifier: AfR16) [25 (link)]; anti-XCAP-H (in-house identifier: AfR20) [25 (link)]; anti-pT1314-XCAP-D2 (in-house identifier: AfR42-P) [25 (link)]; anti-pT1348-XCAP-D2 (in-house identifier: AfR44-P) [25 (link)];anti-pT1353-XCAP-D2 (in-house identifier: AfR46-P) [25 (link)]; anti-hCAP-D2 (in-house identifier: AfR51-5L) [34 (link)]; anti-pT1339-hCAP-D2 (in-house identifier: AfR173-4P) [32 (link)]; anti-pT1384-hCAP-D2 (in-house identifier: AfR175-4P) [32 (link)]; anti-FLAG M2 (Sigma, F-1804 [RRID: AB_262044]); anti-PSTAIR (Abcam, ab10345, [RRID: AB_297080]); anti-pT161-Cdk1 (Cell Signaling Technology, 9114, [RRID: AB_2074652]). Secondary antibodies used in the current study were as follows: horseradish peroxidase-conjugated anti-rabbit IgG (Vector Laboratories, PI-1000 [RRID: AB_2336198]); horseradish peroxidase-conjugated anti-mouse IgG (Vector Laboratories, PI-2000 [RRID: AB_2336198]); IRDye 680RD goat anti-rabbit IgG (LI-COR Biosciences, 925–68071 [RRID: AB_2721181]); IRDye 800CW goat anti-mouse IgG (LI-COR Biosciences, 925–32210[RRID: AB_2687825]).
4
Gal-1 Protein Expression Analysis
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Infected and non-infected HL–1 cells were washed with PBS, harvested and homogenized in ice-cold lysis buffer (10 mM HEPES, 2 mM EDTA, 150 mM NaCl 150, 0.1% NP40) in the presence of a protease inhibitor kit (Complete Mini EDTA-free, Roche, Germany). After protein quantification by Bradford reagent (Sigma-Aldrich, St. Louis, MO, USA), equal amount of protein (60 μg per lane) was resolved on a 15% SDS-PAGE, transferred to nitrocellulose membranes and then immunoblotted with a rabbit anti-Gal–1 polyclonal Ab (dilution 1:3,000) or a mouse monoclonal Ab for β-actin (BD Pharmingen, Chicago, IL, USA) as a loading control. Blots were then incubated with horseradish peroxidase-conjugated anti-rabbit IgG (Vector Labs, UK) or horseradish peroxidase-conjugated anti-mouse IgG (Sigma-Aldrich, St. Louis, MO, USA). Immunoblots were visualized with the Immobilon chemiluminescent horseradish peroxidase substrate (Millipore, Billerica, MA) according to manufacturer’s instructions. The bands were scanned and quantified using ImageJ software (version 1.410).
5
Western Blot Analysis of DMRTA2 Protein
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Cells were lysed in the buffer containing phosphatase and protease inhibitors (20 mM Tris HCl, pH 6.8, 137 mM sodium chloride, 25 mM β-glycerophosphate, 2 mM sodium pyrophosphate, 2 mM EDTA, 1 mM sodium orthovanadate, 1% Triton X-100, 10% glycerol, 5 µg/mL leupeptin, 5 µg/mL aprotinin, 2 mM benzamidine, 0.5 mM DTT, 1 mM PMSF). The protein concentration was determined with the Pierce BCA Protein Assay Kit (Thermo Scientific). Protein extracts were separated on SDS-PAGE before electrophoretic transfer onto a nitrocellulose membrane (Amersham Biosciences, Germany) as described [23 (link)]. After blocking with 5% non-fat milk in TBS-T (Tris-buffered saline pH 7.6/0.15% Tween 20) the membranes were incubated with primary antibody recognizing DMRTA2 (Abcam #ab156244) diluted in a TBS-T overnight at 4 °C and then with horseradish peroxidase-conjugated anti-rabbit IgG (#PI-1000, Vector Laboratories) for one hour at RT. Immunocomplexes were visualized by using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermofisher Scientific). The membranes were stripped and re-probed with horseradish peroxidase-conjugated anti-β-Actin antibody (Sigma-Aldrich) to verify total protein loading.
6
Quantifying UCH-L1 and UCH-L3 Protein Expression
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Tissue extracts or cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) using 12.5% gel for UCH-L1 protein. After being separated
by electrophoresis, proteins were transferred to a polyvinylidene fluoride (PVDF) membrane
and blocked with 5% nonfat dry milk in PBS plus 1% Tween 20 (PBST) for 1 h at room
temperature. The membranes were incubated with anti-UCH-L1 antibody (1:20,000),
anti-UCH-L3 antibody (1:1,000) or anti-β-actin antibody (1:20,000) as an internal control
overnight at 4°C. Then, the membranes were subsequently incubated with horseradish
peroxidase-conjugated anti-rabbit IgG (1:10,000) or horseradish peroxidase-conjugated
anti-mouse IgG (1:10,000) (Vector Laboratories) for 1 h at room temperature.
Immunoreactions were visualized by ECL plus (GE Healthcare, Piscataway, NJ, USA) and were
detected using a CCD camera system (LAS-4000, Fujifilm, Tokyo, Japan).
gel electrophoresis (SDS-PAGE) using 12.5% gel for UCH-L1 protein. After being separated
by electrophoresis, proteins were transferred to a polyvinylidene fluoride (PVDF) membrane
and blocked with 5% nonfat dry milk in PBS plus 1% Tween 20 (PBST) for 1 h at room
temperature. The membranes were incubated with anti-UCH-L1 antibody (1:20,000),
anti-UCH-L3 antibody (1:1,000) or anti-β-actin antibody (1:20,000) as an internal control
overnight at 4°C. Then, the membranes were subsequently incubated with horseradish
peroxidase-conjugated anti-rabbit IgG (1:10,000) or horseradish peroxidase-conjugated
anti-mouse IgG (1:10,000) (Vector Laboratories) for 1 h at room temperature.
Immunoreactions were visualized by ECL plus (GE Healthcare, Piscataway, NJ, USA) and were
detected using a CCD camera system (LAS-4000, Fujifilm, Tokyo, Japan).
7
Western Blot Protein Detection
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Whole-cell protein extracts were separated on SDS-PAGE before electrophoretic transfer onto a nitrocellulose membrane (Amersham Biosciences). The membranes were incubated with primary antibodies diluted in a blocking buffer overnight and then with relevant secondary antibodies for one hour. Antibodies recognizing LRP6 (diluted 1:1000) was purchased from Abcam, tRFP (diluted 1:2000) from Evrogen, β-Actin antibody (diluted 1:2000) from Oncogene Research Products, horseradish peroxidase-conjugated anti-rabbit IgG (diluted 1:2000) from Vector Laboratories. Immunocomplexes were visualized by using ECL (Amersham). The molecular weight of proteins was estimated with pre-stained protein markers (ThermoScientific). Band intensities were determined by densitometric analysis of immunoblots with Molecular Imager FX and Quantity One software (BioRad).
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