SpecificityThe antibody is calibrated against a spectrum of antigens to assure hapten selectivity.
No measurable cross-reactivity (<1:1000) was detected against alanine in peptides or proteins.
Fixed tissue cross-reactivity was tested with known targets at the recommended dilution.
No measurable glutaraldehyde-fixed tissue cross-reactivity (<1:1000) was detected against D/L-aspartate, D/L-arginine, agmatine, guanidine, gamma-aminobutyrate, L-citrulline, L-cysteine, D/L-glutamate, D/L-glutamine, glutathione, glycine, L-lysine, L-ornithine, L-serine, taurine, L-threonine, L-tryptophan, L-tyrosine.
Significant cross-reactivity (1:10) was detected against beta-alanine.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application notesThis antibody is recommended for thin section post-embedding endogenous content mapping by Light and Electron microscopy immunostaining via High Performance Immunocytochemistry (HPI) (see "Protocols" tab above)
Recommended user dilution: 1/100
True Dilution at User Dilution: 1/8000
High-performance thin section immunostaining using silver-intensified immunogold or fluorescence detection.
Enhanced detection is possible using streptavidin detection.
EM applications with 10-40 nm gold GAR IgGs or gold streptavidin.
OPTIMAL FIXATION: 0.1-2.5% glutaraldehyde,1% formaldehyde using HPI protocol (The antisera targets the glutaraldehyde conjugate of the hapten).
MINIMAL GLUTARALDEHYDE: 0.1% using HPI
The hapten is osmium tolerant (deosmication required), therefore the antisera can be used on conventional post-embedding electron microscope immunostaining.
All procedures may be carried out at room temperature. Exact dilutions for all applications cannot be predicted, but it is unlikely that deviations from the calibrated levels will be needed. Dilutions are optimized for antigen detection over a 2 log unit range.
The product is optimized for HPI/EHPI with gold or fluorescence detection using etched plastic sections. Filter diluted reagents with 0.2 mm syringe filters before use on EM grids. Enzyme-linked visualizations can be used but will compress the signal dynamic range and are less sensitive.
Use with frozen or vibratome sections is possible but will not yield optimal images as IgGs penetrate aldehyde cross-linked tissue poorly and most amino acids are present at such high levels that prozone effects occur. Use in whole mounts is not recommended for similar reasons.