WB; IH, Frozen; IF,Frozen; IH(P), Paraffin embedded; IF; IP; ELISA The biotinylated MOAB-2 antibody has been tested by IHC (1:500 - 1:2,000 dilution) and is also expected to work in applications validated for the unlabelled antibody (M-1586-100) at same or higher dilutions: Western Blotting (WB), Immunohistochemistry (IHC), Immunohistochemistry/paraffin embedded IHC(P), Immunoprecipitation (IP), Immunofluorescence (IF), ELISA.
Western Blotting:
MOAB-2 has been tested in WB using purified synthetic beta-amyloid preparations and from transgenic mouse brain formic acid extracts (see Figure 1). Formic acid extraction/concentration is required for western blot detection from extracts. Suggested dilution of 1:2000-1:5,000 for WB, standard ECL detection systems.
Tissue samples for the detection of beta-amyloid should be prepared as detailed in Youmans KL et al., 2011 (Journal of Neuroscience Methods 196: 51-59) for best results. Detection of beta-amyloid 40/42 in direct westerns can be difficult; Dot-blots of prepared samples are recommended as detailed in Youmans KL et al., 2012.
Immunohistochemistry:
Suggested dilution for biotinylated MOAB-2 in IHC is 1:500-1:2,000. Fresh frozen, 4% paraformaldehyde fixed frozen, or formalin fixed paraffin embedded tissues are all suitable. Antigen retrieval is required in fixed tissues for optimal staining.
Antibody was tested on 4% paraformaldehyde/0.1% glutaraldehyde fixed frozen tissue from 3xTg and 5xFAD mice. MOAB-2 antibody detects intraneuronal and extracellular beta-amyloid in IHC and does not detect APP (Youmans KL et al., 2012).
The antibody also reacts with archival formalin-fixed, paraffin-embedded tissue samples with antigen Heat Induced Epitope Retrieval (HIER). Recommended buffer for HIER is citrate, pH 6.0. Signal was weak without antigen retrieval. Immunoreactivity was observed in intraneural-amyloid deposition (plaque) in Alzheimer's brain. MOAB-2 was found to be extremely clean and with an excellent signal to noise ratio with no neuro-cellular diffusive staining.
In addition, MOAB-2 demonstrated no significant differences in A-beta detection using paraffin fixed, free-floating sections (Youmans KL et al., 2012). Formic acid (FA) treatment resulted in optimal detection of both intraneuronal and extracellular A-beta compared to without FA (incubated in 88% FA 8 min, Youmans KL et al., 2012). Free floating tissue sections were permeabilized in TBS containing 0.25% Triton X-100 (TBSX; 3 x 10 min), blocked with 3% horse serum in TBSX (3 x 10 min) followed by 1% horse serum in TBSX (2 x10 min) and incubated with appropriate primary antibodies diluted in TBSX containing 1% horse serum overnight. See Youmans KL et al., 2012, for full IHC(P) protocol and method details.
Immunofluorescence:
For IF, suggested dilution is 1:100-1:500. The antibody was tested on 4% PFA fixed frozen tissue. Fixed tissues were washed in TBS (3 x 10 min), then incubated in 88% FA (8 min), and then permeabilized in TBSX (3 x 10 min), and blocked in TBSX containing 5% bovine serum albumin (BSA; 1 hr). Sections were subsequently incubated with appropriate primary antibodies diluted in TBSX containing 2% BSA overnight on an oscillatory rotator. Detection was via fluorescently labelled absorbed secondary antibodies (Youmans KL et al., 2012).
Immunoprecipitation:
For IP, the suggested dilution is 1:200 to 1:1,000 for labelled beta-amyloid using SA-coated beads as the capture vehicle, similar to the protocols employed by Youmans KL et al., 2012.
ELISA:
In an ELISA, a dilution of 1:50-1:1,000 is suggested. The antibody has been tested in ELISAs on synthetic beta-amyloid and tissue homogenates from beta-amyloid-Tg mice.
Biosensis recommends optimal dilutions/concentrations should be determined by the end user for all applications. Dilutions provided are only meant to serve as a basic guide.
免疫原
Recombinant human amyloid beta protein 42 (AB42): DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA
来源宿主
小鼠
反应性
人, 大鼠, other species not yet tested. By Dot Blot, MOAB-2 detected 大鼠 AB40, 人 AB40, albeit with less affinity than for AB42 (Youmans KL et al., 2012).