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Peroxidase conjugated anti flag antibody

Manufactured by Merck Group
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The Peroxidase-conjugated anti-FLAG antibody is a laboratory reagent used for the detection and identification of proteins tagged with the FLAG peptide epitope. The antibody is conjugated to the enzyme horseradish peroxidase, which allows for colorimetric or chemiluminescent detection of the target protein in various applications, such as Western blotting, immunoprecipitation, and enzyme-linked immunosorbent assays (ELISA).

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Lab products found in correlation

Envision multiwell reader, by PerkinElmer (4 mentions) Baff fc, by Sino Biological (2 mentions) April, by Merck Group (2 mentions) Human serum albumin (hsa), by Merck Group (2 mentions) Recombinant baff, by Sino Biological (2 mentions) Maxisorp, by Thermo Fisher Scientific (2 mentions) Peroxidase conjugated anti human fc, by Merck Group (1 mentions)

6 protocols using peroxidase conjugated anti flag antibody

1

Epitope Binning and Affinity Characterization of VNARs

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For epitope binning, 96-well Maxisorp plates were coated at 1 μg/ml with either hBAFF-Fc and washed with blocking buffer. Selected clones were grown in a 96 deep-well format in auto-induction medium and periplasmic extracts were pre-blocked in the presence of a competitor VNAR-Fc molecule at 2 μM final concentration. The extracts were then exposed to the coated surface and bound VNARs were detected using a peroxidase-conjugated anti-FLAG antibody (Sigma).
The biochemical EC50 of selected clones was determined by serially diluting purified monomeric VNARs in blocking buffer and binding to preblocked 96-well plates coated at 1 μg/ml with hBAFF-Fc. After washing in PBS-0.1% Tween, bound VNARs were detected using a peroxidase-conjugated anti-FLAG antibody (Sigma). The biochemical IC50 of selected clones on all three BAFF receptors was determined by serially diluting purified monomeric VNARs in blocking buffer containing 1 nM hBAFF-Fc in the case of BR3 or 0.5 nM in the case of TACI and BCMA. The pre-blocked proteins were then exposed to 96-well plates coated at 1 μg/ml with extra-cellular domains of BR3, TACI or BCMA. After washing in PBS-0.1% Tween, bound hBAFF was detected using a peroxidase-conjugated anti-human Fc. Absorbance at 450 nm was recorded and EC50/IC50 values were calculated by non-linear regression using GraphPad Prism®.
Häsler J., Flajnik M.F., Williams G., Walsh F.S, & Rutkowski J.L. (2016). VNAR single-domain antibodies specific for BAFF inhibit B cell development by molecular mimicry. Molecular immunology, 75, 28-37.
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2

Screening BAFF-Blocking Antibody Clones

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Example 7

Clones blocking the interaction between BAFF and its three receptors were tested for their ability to cross-react with BAFF's closest related protein, APRIL. Clones were grown in a 96 well format, periplasmic fraction was extracted by osmotic shock as previously described and directly used in a binding ELISA. Nunc Maxisorp 96 well plates were coated at 1 μg/mL with either BAFF-Fc (Sino Biological), HSA (Sigma), or APRIL (Sigma) and periplasmic fraction, pre-blocked in PBS-0.1% Tween+2.5% Milk, was exposed to the coated surface. After washing in PBS-0.1% Tween, bound VNARs were detected using a peroxidase-conjugated anti-FLAG antibody (Sigma). Absorbance at 450 nm was recorded using an Envision multiwell reader (Perkin Elmer). As shown in FIG. 13, with the exception of B07 which showed a slight binding to APRIL, all the blocker clones appeared specific to BAFF.

US10435474B2. Baff selective binding compounds and related methods (2019-10-08). Ossianix, Inc. [US]. Inventors: Julien Häsler [GB], Julia Lynn Rutkowski [US].
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3

Competitive Epitope Mapping of Anti-BAFF VNARs

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Example 8

In order to group the blocking clones into different categories based on the epitope that each one recognizes on the BAFF proteins, Nunc Maxisorp 96 well plates were coated at 1 μg/mL with recombinant BAFF (Sino biologicals). Clones were grown in a 96 well format and periplasmic fraction was extracted by osmotic shock as previously described. The periplasmic fraction was then pre-blocked in PBS-0.1% Tween+2.5% Milk in the presence of a competitor VNAR-Fc molecule at 2 μM final concentration. Two anti-BAFF VNAR-Fcs were used in this assay (A07, B07) as well as a negative control lysozyme-binding VNAR (5A7). The pre-blocked fraction was then exposed to the coated surface, and after washing in PBS-0.1% Tween, bound VNARs were detected using a peroxidase-conjugated anti-FLAG antibody (Sigma). Absorbance at 450 nm was recorded using an Envision multiwell reader (Perkin Elmer). The results are shown in FIG. 14. Results showed that the binding of each clone to BAFF was competed by both A07 and B07, revealing that all the clones of the selected panel of blockers target the same (or at least an overlapping) epitope on BAFF.

US10435474B2. Baff selective binding compounds and related methods (2019-10-08). Ossianix, Inc. [US]. Inventors: Julien Häsler [GB], Julia Lynn Rutkowski [US].
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4

Epitope Mapping of BAFF Binder Clones

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Example 8

In order to group the blocking clones into different categories based on the epitope that each one recognizes on the BAFF proteins, Nunc Maxisorp 96 well plates were coated at 1 μg/mL with recombinant BAFF (Sino biologicals). Clones were grown in a 96 well format and periplasmic fraction was extracted by osmotic shock as previously described. The periplasmic fraction was then pre-blocked in PBS-0.1% Tween+2.5% Milk in the presence of a competitor VNAR-Fc molecule at 2 μM final concentration. Two anti-BAFF VNAR-Fcs were used in this assay (A07, B07) as well as a negative control lysozyme-binding VNAR (5A7). The pre-blocked fraction was then exposed to the coated surface, and after washing in PBS-0.1% Tween, bound VNARs were detected using a peroxidase-conjugated anti-FLAG antibody (Sigma). Absorbance at 450 nm was recorded using an Envision multiwell reader (Perkin Elmer). The results are shown in FIG. 14. Results showed that the binding of each clone to BAFF was competed by both A07 and B07, revealing that all the clones of the selected panel of blockers target the same (or at least an overlapping) epitope on BAFF.

US11267894B2. BAFF selective binding compounds and related methods (2022-03-08). Ossianix, Inc. [US]. Inventors: Julien Häsler [BE], Julia Lynn Rutkowski [US].
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5

VNAR Binding and Blocking ELISA

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Individual clones were grown in 96 deep-well plates in auto-induction medium (EMD Millipore, Germany) and the periplasmic fraction was extracted by osmotic shock as described in (Muller et al., 2012 (link)). For the binding ELISA, Maxisorp plates were coated at 1 μg/ml with either hBAFF-Fc or HSA and periplasmic fraction, in blocking buffer (2.5% non-fat dry milk and 0.1% Tween-20 in PBS) was exposed to the coated surface. After washing in PBS-0.1% Tween, bound VNARs were detected using a peroxidase-conjugated anti-FLAG antibody (Sigma) and absorbance at 450 nm was recorded. Cross-reactivity of selected clones to rhAPRIL (Sigma) was tested using an identical ELISA protocol. For the blocking ELISA, plates were coated at 1 μg/ml with extracellular domains of BR3, TACI, or BCMA (all from Peprotech, USA) and washed with blocking buffer. The pre-blocked periplasmic fraction with the addition of hBAFF-Fc (5 nM in the case of BR3 and 0.5 nM in the case of TACI and BCMA) was then exposed to the coated surface, and bound hBAFF was detected with a peroxidase-conjugated anti-human Fc (Sigma).
Häsler J., Flajnik M.F., Williams G., Walsh F.S, & Rutkowski J.L. (2016). VNAR single-domain antibodies specific for BAFF inhibit B cell development by molecular mimicry. Molecular immunology, 75, 28-37.
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6

Screening Antibody Clones for BAFF Specificity

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Example 7

Clones blocking the interaction between BAFF and its three receptors were tested for their ability to cross-react with BAFF's closest related protein, APRIL. Clones were grown in a 96 well format, periplasmic fraction was extracted by osmotic shock as previously described and directly used in a binding ELISA. Nunc Maxisorp 96 well plates were coated at 1 μg/mL with either BAFF-Fc (Sino Biological), HSA (Sigma), or APRIL (Sigma) and periplasmic fraction, pre-blocked in PBS-0.1% Tween+2.5% Milk, was exposed to the coated surface. After washing in PBS-0.1% Tween, bound VNARs were detected using a peroxidase-conjugated anti-FLAG antibody (Sigma). Absorbance at 450 nm was recorded using an Envision multiwell reader (Perkin Elmer). As shown in FIG. 13, with the exception of B07 which showed a slight binding to APRIL, all the blocker clones appeared specific to BAFF.

US11267894B2. BAFF selective binding compounds and related methods (2022-03-08). Ossianix, Inc. [US]. Inventors: Julien Häsler [BE], Julia Lynn Rutkowski [US].
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