Hrp conjugated anti his antibody

Manufactured by Merck Group

The HRP conjugated anti-His antibody is a laboratory reagent used for the detection and identification of proteins with a histidine (His) tag. It is a secondary antibody that binds to the His-tagged protein and is conjugated with horseradish peroxidase (HRP), enabling colorimetric or chemiluminescent detection.

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Lab products found in correlation

Fiberlite f21 8 x 50y, by Thermo Fisher Scientific (2 mentions) Talon resin, by Takara Bio (2 mentions) Maxisorp immunotubes, by Thermo Fisher Scientific (1 mentions) Pip strip, by Echelon Biosciences (1 mentions) Isopropyl β d 1 thiogalactopyranoside (iptg), by Takara Bio (1 mentions) Talon metal affinity resin, by Takara Bio (1 mentions) Ecl select western blotting detection reagent, by GE Healthcare (1 mentions) Imidazole, by Merck Group (1 mentions)

4 protocols using hrp conjugated anti his antibody

Affinity Purification of His-Tagged Protein Complexes

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Cell pellet was resuspended in buffer (25 mM Tris-HCl pH 8.0, 150 mM
NaCl, 5 mM Imidazole, EDTA free complete protease inhibitor (Roche), lysed by 2
cycles of freeze thaw in liquid nitrogen and the lysate was cleared by
centrifugation at 19,000 g in a Thermo Scientific™ Fiberlite™
F21-8 x 50y fixed-angle rotor for 90 min. The supernatant was mixed with Talon
resin (Clontech), equilibrated in buffer A (25 mM Tris-HCl pH 8.0, 150 mM NaCl,
5 mM Imidazole) and incubated for 1 hour at 4° C. The resin was washed
with 15 column volumes (CV), 10 CV of buffer HS (25 mM Tris-HCl pH 8.0, 1000 mM
NaCl, 5 mM Imidazole), and 15 CV of buffer A before eluting the bound protein
complex with 5 CV of buffer B (25 mM Tris-HCl pH 8.0, 150 mM NaCl, 200 mM
Imidazole). Different samples were analysed by SDS-PAGE analysis and western
blot analysis using HRP conjugated anti-His antibody (Sigma-Aldrich) for TAF5NTD
and anti-TAF6 (primary, received from Laszlo Tora) followed by HRP conjugated
anti-mouse (secondary, Sigma-Aldrich) for TAF6.

Antonova S.V., Haffke M., Corradini E., Mikuciunas M., Low T.Y., Signor L., van Es R.M., Gupta K., Scheer E., Vos H.R., Tora L., Heck A.J., Timmers H.T, & Berger I. (2018). Chaperonin CCT Checkpoint Function in Basal Transcription Factor TFIID Assembly. Nature structural & molecular biology, 25(12), 1119-1127.

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VNAR Library Screening for DLL4 Binders

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VNAR phage display library screening VNAR E4 was isolated from a synthetic VNAR library containing 100 billion unique clones. Four rounds of solid phase, phage display antigen selections were carried out as described previously 18 using MaxiSorp immunotubes (Nunc) coated with 10-4 µg ml -1 rhDLL4 (recombinant human) in PBS pH 7.4. Outputs from each selection round were screened for specificity to human and murine DLL4 (1 µg ml -1 in PBS coating concentration) by monoclonal phage and periplasmic extract ELISAs. Phage binders were detected using HRP-conjugated anti-M13 antibody (Stratech Scientific), and periplasmic protein was detected using an HRP-conjugated anti-His antibody (Sigma-Aldrich).

Leach A ., Smyth P ., Ferguson L ., Steven J ., Greene MK ., Branco CM ., McCann AP ., Porter A ., Barelle CJ , & Scott CJ . (2020). Anti-DLL4 VNAR targeted nanoparticles for targeting of both tumour and tumour associated vasculature. Nanoscale, 12(27).

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Affinity Purification of His-Tagged Protein Complexes

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Purification and Phosphoinositide Binding of Bcas3 Proteins

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Both the His-Dl-bcas3 and His-Dl-bcas3^LAAG plasmids were transformed into E. coli BL21(DE3)pLysS (BioDynamics Laboratory Inc, DS260) and expression was induced with 0.1 mM IPTG (Takara Bio, 9030). Total protein was isolated by sonicating the bacteria in binding buffer (300 mM NaCl, 50 mM sodium phosphate, pH 7.0) containing EDTA-free cOmplete™ protease inhibitors. The his-tagged Bcas3 proteins were purified by incubation with Talon Metal Affinity resin (Clontech, 635501), washing of the resin with 10 mM imidazole (Merck, 137098) in binding buffer and elution with 200 mM imidazole in binding buffer with protease inhibitors. The eluate was concentrated using Amicon Ultracell 30K (Merck Millipore, UFC903008) and dialyzed with TBS (50 mM Tris-Cl, 150 mM NaCl, pH 7.5). PIP strips (Echelon Biosciences, P-6001) were incubated with approximately 200 ng of His-Dl-Bcas3^LKRG or His-Dl-Bcas3^LAAG in TBS containing 0.05% Tween 20 (VWR, 437082Q) and 3% BSA (Sigma-Aldrich, A7906), and after washing with HRP-conjugated anti-His antibody (Sigma-Aldrich, A7058), followed by HRP detection using ECL Select Western Blotting Detection Reagent (GE Healthcare, RPN223).

Yamada Y, & Schaap P. (2020). The proppin Bcas3 and its interactor KinkyA localize to the early phagophore and regulate autophagy. Autophagy, 17(3), 640-655.

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