Primary antibodies used were rabbit anti-brevican “neo” (Rb399) (custom-made; rat neo-epitope QEAVESE; 1:1000) [42 (link)], polyclonal sheep anti-human/rat brevican (AF4009, R&D Systems, Minneapolis, MN, USA, 1:2000), polyclonal sheep anti-rat/mouse neurocan (AF5800, R&D Systems, Minneapolis, MN, USA, 1:1000), polyclonal goat anti-human HAPLN1 (AF2608, R&D Systems, Minneapolis, MN, USA, 1:200), polyclonal rabbit anti-aggrecan (AB1031, Sigma-Aldrich/Merck KGaA, Darmstadt, Germany, 1:500), and the antibodies from the ELISA kits (see below). Secondary antibodies used were peroxidase-conjugated AffiniPure donkey anti-rabbit IgG (H + L; 1:2000 for aggrecan; 1:20,000 for brevican neo), peroxidase-conjugated AffiniPure donkey anti-sheep IgG (H + L; 1:5000), and peroxidase-conjugated AffiniPure donkey anti-goat IgG (H + L; 1:2000); (all Jackson ImmunoResearch, Cambridgeshire, UK).
Af4009
Manufactured by R&D Systems
Sourced in United States
AF4009 is a laboratory equipment designed for DNA/RNA isolation and purification. It utilizes a proprietary technology to efficiently extract and concentrate targeted nucleic acid molecules from a variety of sample types. The core function of AF4009 is to provide a streamlined and reliable method for the isolation and purification of nucleic acids for downstream applications such as PCR, sequencing, and molecular analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ab1031, by Merck Group (2 mentions)
Af5800, by R&D Systems (2 mentions)
Peroxidase conjugated affinipure donkey anti sheep igg, by Jackson ImmunoResearch (1 mentions)
Peroxidase conjugated affinipure donkey anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 594 conjugated streptavidin, by Thermo Fisher Scientific (1 mentions)
Mab5406, by Merck Group (1 mentions)
Texas red conjugated goat anti rabbit, by Vector Laboratories (1 mentions)
B 1355, by Vector Laboratories (1 mentions)
Alexa fluor 488 conjugated donkey anti sheep igg, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated anti mouse igm sc 2082, by Santa Cruz Biotechnology (1 mentions)
Af3865, by R&D Systems (1 mentions)
Affinipure donkey anti rabbit igg h l, by Jackson ImmunoResearch (1 mentions)
3 protocols using af4009
1
Antibody Detection of Chondroitin Proteoglycans
2
Immunolabeling of Extracellular Matrix Proteins
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The following lectins and primary antibodies were used for staining: biotinylated WFA (B-1355, Vector Laboratories; 1:200), mouse anti-PV (clone PARV-19, P3088; Sigma-Aldrich Japan, Tokyo, Japan; 1:1000), mouse anti-aggrecan (Cat-315; MAB1581, MERCK; 1:1000), rabbit anti-aggrecan (AB1031, Millipore, Tokyo, Japan; 1:200), goat anti-tenascin-R (AF3865, R&D Systems, Minneapolis, MN, USA; 1:200), sheep anti-brevican (AF4009, R&D Systems; 1:200), and mouse anti-glutamate decarboxylase (GAD67) (clone 1G10.2, MAB5406; Millipore, Bedford, MA; 1:1000).
The following secondary antibodies were used for visualization: Alexa Fluor 488-conjugated goat anti-mouse immunoglobulin (Ig)G (ab150113; Abcam, Cambridge, MA; 1:1000), FITC-conjugated anti-mouse IgM (sc-2082, Santa Cruz Biotechnology, Santa Cruz, CA, 1:1000), Texas Red-conjugated goat anti-rabbit (TI-1000; Vector Laboratories, Funakoshi Co., Tokyo, Japan), DyLight488-conjugated horse anti-goat IgG (DI-3088; Vector Laboratories; 1:500), Alexa Fluor 488-conjugated donkey anti-sheep IgG (A-11015; Thermo Fisher Scientific, Kanagawa, Japan; 1:1000), and streptavidin-conjugated Alexa Fluor 594 (S11227, Thermo Fisher Scientific; 1:1000).
The following secondary antibodies were used for visualization: Alexa Fluor 488-conjugated goat anti-mouse immunoglobulin (Ig)G (ab150113; Abcam, Cambridge, MA; 1:1000), FITC-conjugated anti-mouse IgM (sc-2082, Santa Cruz Biotechnology, Santa Cruz, CA, 1:1000), Texas Red-conjugated goat anti-rabbit (TI-1000; Vector Laboratories, Funakoshi Co., Tokyo, Japan), DyLight488-conjugated horse anti-goat IgG (DI-3088; Vector Laboratories; 1:500), Alexa Fluor 488-conjugated donkey anti-sheep IgG (A-11015; Thermo Fisher Scientific, Kanagawa, Japan; 1:1000), and streptavidin-conjugated Alexa Fluor 594 (S11227, Thermo Fisher Scientific; 1:1000).
3
Brevican Neo-Epitope Detection in CSF
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Following primary antibodies were used: rabbit anti-brevican “neo” (Rb399) (custom-made; rat neo-epitope QEAVESE; see Valenzuela et al., 2014 (link); 1:500), polyclonal sheep anti-human/rat brevican (AF4009, R&D Systems, Minneapolis, United States, 1:1000), polyclonal sheep anti-rat/mouse neurocan (AF5800, R&D Systems, Minneapolis, United States, 1:1000), and the antibodies from the ELISA kits (see above).
To prove if the neo-antibody, which was originally produced against the neo-epitope of the rat N-terminal brevican fragment, is also detecting the human brevican fragment neo-epitope EATESE we performed a competition assay with pre-incubation of the primary antibody with a 100-fold molar excess of the peptide Biotin-Ahx-CGQEAVESE containing the immunogen for 1 h at RT. As shown inSupplementary Figure 1 , the 60 kDa band detected by this antibody in human CSF samples is clearly diminished in the peptide competition condition, demonstrating its specificity.
As secondary antibodies we used peroxidase-conjugated AffiniPure donkey anti-rabbit IgG (H + L) and peroxidase-conjugated AffiniPure donkey anti-sheep IgG (H + L) (Jackson ImmunoResearch, Cambridgeshire, United Kingdom, 1:2000).
To prove if the neo-antibody, which was originally produced against the neo-epitope of the rat N-terminal brevican fragment, is also detecting the human brevican fragment neo-epitope EATESE we performed a competition assay with pre-incubation of the primary antibody with a 100-fold molar excess of the peptide Biotin-Ahx-CGQEAVESE containing the immunogen for 1 h at RT. As shown in
As secondary antibodies we used peroxidase-conjugated AffiniPure donkey anti-rabbit IgG (H + L) and peroxidase-conjugated AffiniPure donkey anti-sheep IgG (H + L) (Jackson ImmunoResearch, Cambridgeshire, United Kingdom, 1:2000).
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