Nanogold particles is a 1.4 nm diameter gold compound which can be covalently and
specifically linked to an antibody or to a hinge thiol on Fab' or IgG. These substantially
smaller antibody-Nanogold probes reach more antigens and provide better labeling
and stability compared to colloidal gold preparation. They can be viewed directly
in TEM without silver enhancement or developed with silver to any appropriate size
for enhanced visibility with other counter stains.
Bio-Synthesis can custom conjugate 1.4 nm Nanogold-antibody conjugates using your
own primary antibody (igG or Fab'), protein's peptide, lectins, and other molecules.
Contact us with your specific needs.
Features of Nanogold Antibody Conjugates
- Excellent stability compared to colloidal gold -antibody preparation
- Smallest commercially available gold immunoprobes
- Unparalleled penetration of conjugates up to 40µm
- Extremely uniform 1.4nm diameter gold label
- Close to one Nanogold® particle to one Fab' (or IgG)
- Low non-specific affinity gives minimal background
- Ultrasensitivity with silver enhancement: 0.1pg of antigen
detection on immunoblots
- Label at specific sites which do not obstruct antigen binding
region
- High stability and long shelf life up to a years
- Conjugates are stable to a wide range of pH and ionic strengths
Conjugation Chemistries
Standard immobilization of antibodies using modified/activated or native solid supports,
maleimide activated nanogold can be used with a variety of homo- or heterobifunctional
crosslinkers, or directly label with RNA or glycoprotein selectively through thiols,
or modified sulfo-NHS-Nanogold with primary aliphatic amines (N-terminal or lysine
residues).
Quality Control Criteria
The custom prepared product is quality controlled by Bio-Synthesis according to
our in-house standards with respect to size distribution and clustering. In agreement
with the principal investigator the binding substance's specific quality control
criteria are determined. These are tailored for each individual assignment. The
custom conjugate is approved only when both sets of quality control criteria have
been met.
Custom Formulation
Our conjugation is based on a mixed short length polymer cross linked covalent bonded
technique. We require ligands with either a primary amine or are biotinylated.
Prices
Due to the nature of each project, a written quotation will be made stating cost
and time required after receipt of the antibody sample.
Delivery
Labeled products will be supplied as 1 ml, containing 80 µg of labeled antibody.
Products will be shipped in 20 mM sodium phosphate solution, pH 7.4, with 150 mM
sodium chloride, 0.1% bovine serum albumin and 0.05% sodium azide as preservative
(expiration date 12 months from packing), unless you specify otherwise (for example,
conjugates may be supplied without bovine serum albumin). Custom labeled conjugates
can be supplied in the 1 mL size only; we cannot accept orders for smaller sizes.
This conjugation is guaranteed and based on dynamic light scattering (DLS) results
for both size and charge. Surface coverage, reactivity, and activity of the conjugation
are unmeasured. Conjugations take 1-2 weeks. All shipments are sent Fed Ex Standard
Overnight for domestic delivery.
No shipments on Fridays.
Sample Submission Guideline
As a rule, Bio-Synthesis will only accept pure substances as starting material,
either lyophilized or dissolved in a medium of known chemical composition. Interfering
substances must not be present. Non-commercial antibodies supplied by the customer
should be affinity purified. Please provide gel electrophoresis data along with
your antibody. Commercially available antibodies can be supplied by customers or
ordered through Bio-Synthesis. We can assist in antibody modification or fragmentation
prior to any immobilization reaction.
Antibody Supplied by Customers
We can also custom label your monoclonal IgG molecules, or prepare labeled Fab'
fragments. Fab' fragments can be labeled if they are supplied as F(ab')2 fragments;
where IgG is supplied, labeled Fab' fragments can only be produced only for the
IgG1 and IgG2a subclasses, not from IgG2b or IgG3 monoclonal antibodies. We recommend
that you consider using our labeling reagents, monomaleimido Nanogold® and monomaleimido
undecagold, before placing a custom synthesis order; these reagents will label antibodies
using the same reaction in the same manner as our process for preparing catalog
conjugates.
Amount of Antibody Required:
- For labeling of IgG molecules: 500 µg
of antibody is required.
- For preparation of labeled Fab' fragments room F(ab')2
fragments: 500 mg of F(ab')2 fragments is required.
- For preparation of labeled Fab' fragments from IgG
molecules: 2 mg of IgG is required.
The following data are particularly useful:
Molecular weight
All of our labeling reactions use specific molar ratios of gold to probe. We need
the molecular weight to calculate how much gold labeling reagent we will need, how
much of the probe we will need to use for labeling, and also which product isolation
protocol to use.
Optical density or UV/visible absorption data
The extinction coefficients of undecagold, Nanogold® and FluoroNanogold at specific
wavelengths have been accurately determined, and these values allow us to determine
how successfully your probe has been labeled. If you supply or refer us to specific
values for your probe, we can provide you with the exact ratio of gold cluster label
to probe in the product.
Molecular structure and functional groups
Ideally, targeted biomolecules should be labeled at a unique reactive group, remote
from the substrate binding region, and we try to identify suitable sites for labeling,
such as a unique thiol or a terminal primary amine. Some idea of the overall structure
of your molecule and which regions are thought to bind to the target would be very
helpful in selecting a labeling site. Thiols, primary amines, carboxylates and aldehydes
may all be labeled. Labeling through hydroxyls or cis-1,2-dihydroxy groups is also
possible; other groups may require extensive synthetic modifications which will
increase the cost and synthesis time considerably.
References for labeling with other tags
We can often obtain a good idea about which labeling approaches might work from
related literature articles. A paper which describes a successful labeling with
a fluorophore, for example, is a good starting point for evaluating potential gold-labeling
strategies. Our gold labels are conjugated using similar cross-linking reactions
and have similar site requirements. Data on properties such as solubility and compatibility
with various solvents are also useful because small molecules can sometimes alter
the solubility properties of gold clusters significantly. Knowing an alternative
solvent beforehand saves time spent solubility testing later.