rev:  August 20, 2003

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  APOLIPOPROTEIN E 

ANTIGEN

RDI Division of Fitzgerald Industries Intl  offers a wide line of  antibodies. Since no one antibody works best for all applications (neutralization, blotting, ELISA, etc), we offer many different types of antibodies to help solve this problem. Please inquire for other applications or types of antibodies not listed below.


see also new E Coli produced versions
Recombinant Human Apolipoproteins (E2, E3 and E4)

Isoform        Product#                      50ug               0.5mg

Apo E2        RDI-APOE2-AG        $344.00          $3125.00

Apo E3        RDI-APOE3-AG        $344.00          $3125.00

Apo E4        RDI-APOE4-AG        $344.00          $3125.00

Source: Recombinant human apolipoproteins E2, E3 and E4 are produced by baculovirus-mediated expression in the Spodoptera frugiperda (Sf) insect cell line.

Concentration: see batch sheets approx 0.48mg/ml in 0.7M ammonium bicarbonate


Activity: competes with iodinated human low density lipoprotein for binding to the human apo B/E (LDL) receptor and also binds  to B amyloid peptides in a soluble binding assay.

Physicalproperties: human rApoE is soluble in aqueous solutions. At concnetrations greater than 1.0mg/ml, there may be no free monomer due to self-association common to the amphipathic apoproteins. Two dimensional electrophoresis performed according to the method of O;Farrell indictaes a pI for the primary isoform of approx 6.25 for rApo E2, 6.35 for rApo E3 and approx 6.55-6.7 for rApo E4. Minor acidic isofroms are present and appear to be due to glycosylation and deamination differences.


MW: approx 34 Kda by SDS-Page, with concentrations of 2% SDS, 1.2M UREA, 10% B-mercapoethanol or 10mM DTT.

Blotting: reacts with many monoclonal and polyclonal antibodies


Background: The discovery by Corder and colleagues that apolipoprotein E (apo E) isoforms are associated with the onset of Alzheimer's disease in late onset families has renewed the interest in the function of this important member of the apolipoprotein family. ApoE has also been known to be a ligand for the low density lipoprotein (LDL) receptor and, as early as 1986, researchers at the J. David Gladstone Institutes (San Francisco, Ca) showed that APO E was an important player in nerve degeneration and regeneration. Ongoing research points to new roles for APO E in neuron function and suggests the potential for the development of new therapeutics against crippling diseases in the neurological sciences. These recombinant apolipoproteins retain their full biological activity,allowing for the study of interactions with beta-amyloid, tau, and the LDL- receptor.
-copyright by ownner(s)

For In Vitro Research Use Only-Not for use in or on humans or animals


What are the differences between Apo E2, E3, and E4?

The amino acid sequences of the Apo E isoforms differ at two positions, #112 and #158. These differences are listed below:

Apo E2: Cys-112 and Cys-158

Apo E3: Cys-112 and Arg-158

Apo E4: Arg-112 and Arg-158

What is the source of the Apo E DNA clones?

The DNA clones used for the expression of the human recombinant Apo Es were derived from clones that were originally isolated in the 1980s by prominent Apo E researchers at the J. David Gladstone Institutes.

Are the Apo Es expressed and purified as fusion proteins?

No. They are expressed and purified in their native conformation.

What type of expression system is used to produce the recombinant human Apo Es?

The isoforms are produced by baculovirus-mediated expression in Spodoptera frugiperda (Sf) cells.

What types of post-translational modifications occur with the Apo E isoforms?

Two-dimensional gels reveal a complicated isoform pattern, very similar to that seen with human serum Apo E. Along with the primary recombinant Apo E band, three additional bands occur at more acidic isoelectric points (pI), representing sialylated forms of Apo E. In addition, each band appears as a dimer, the lower molecular weight partner representing the deaminated form of the protein. The relative abundance of each recombinant isoform differs in respect to serum Apo E. With the recombinant Apo Es, the most basic isoform accounts for approximately 90% of the total protein. With serum Apo E, the most basic isoform may only account for 60% of the total protein, with the acidic isoforms contributing 10-20% each. The isoelectric point of the recombinant E2, E3, and E4 differ because of the amino acid substitutions that are unique to each protein. The isoforms have isoelectric points of approximately 6.25 (Apo E2), 6.35 (Apo E3), 6.7 (Apo E4).

How are the recombinant human Apo Es purified?

Each isoform is purified by a scheme that includes affinity chromatography. THis purification scheme does not include steps that involve delipidation or denaturation of the Apo E. Realize, however, that they do not contain the amount of lipid normally associated with Apo E in vivo.

What specific conditions are required for the storage and handling of the Apo Es?

Each isoform is stored in 0.7 M ammonium bicarbonate at -20 to -80°C. Under these conditions, Apo E is very stable over time. However, Apo E has a tendency to aggregate and come out of solution after several freeze-thaw cycles. We recommend that customers limit the number of freeze-thaw cycles to which the ApoEs are exposed.

Does the beta-amyloid protein (bAP) bind to Apo E?

Some studies suggest that the method used to purify Apo E affects its avidity for bAP.(1) The purification scheme does not include delipidation or denaturation steps, and our customers consistently report that all three of our recombinant Apo Es bind well to bAP using the method described by Strittmatter.(2)

Does tau bind to Apo E?

Many researchers have encountered difficulties using gel-shift assays to measure binding between tau, serum Apo E, and recombinant Apo E from bacteria or baculovirus. According to gel-shift assays, tau binds weakly to serum Apo E and undetectably to both recombinant Apo Es. However, some researchers have seen successful binding using an overlay-type of ligand blot assay instead of the gel-shift assay. Using this method, tau binds all three types of Apo Es equally well.(3)

Which Apo Es compete with LDL for LDL-receptor binding?

Recombinant Apo E3 and E4, reconstituted in phospholipid liposomes, are able to compete with iodinated LDL for binding to the classic LDL receptor. Apo E2 cannot compete for binding to the LDL receptor due to its amino acid substitution at position 158. (4)

How do you quantitate and determine the molecular weight of the Apo Es?

-determines the molecular weight of the Apo Es by 2-dimensional electrophoresis and quantitates the proteins using the Bradford method. The 0.7 M ammonium bicarbonate buffer that these recombinant Apo Es are stored in will interfere with most common protein quantitation assays, including Lowry and BCA Assays. However, the buffer is compatible with the Bradford Assay if proper controls are included. To account for the ammonium bicarbonate in each sample tube, add a volume of 0.7 M ammonium bicarbonate that is equal to the volume of Apo E in the sample tubes to each zero blank and standard tube in your Bradford Assay. We recommend using bovine serum albumin as a standard.

How should I perform immunoblotting with the Apo Es?

These recombinant Apo E isoforms react well with many commercially available polyclonal antibodies. Several higher molecular weight bands, representing various multimers of Apo E, also react strongly upon immunoblotting. Also available is a polyclonal antibody that recognizes all three Apo E isoforms (cat#RDI-EAPOEabGX  $375.00/0./4ml) . We have not tested our Apo Es for reactivity with monoclonal antibodies; however, we do know that Apo E electrotransfers well.

How do the Apo Es appear on an SDS-PAGE gel?

The Apo Es migrate on an SDS-PAGE gel at 34,000 Da. We recommend using standard Laemmli sample buffer(5) with the following approximate final concentrations: 0.6% SDS, 1.2 M urea, and 10% betamercaptoethanol (BME). In the absence of urea or BME, the pattern of Apo E monomers and multimers may vary. Apo E stains slowly with Coomassie R-250. We recommend staining minigels for 90 minutes and standard gels overnight.

With a nondenaturing, nonreducing 1-D gel, most of the Apo E remains in the well. Why does this happen?

Apo E will migrate normally on an SDS-PAGE gel. However, under nondenaturing and nonreducing conditions, Apo E can form dimers and multimers because it is an amphipathic protein. To ensure that you have monomers in solution, it may be necessary to include urea or guanidine-HCl in your gel. However, you must be aware of the effect that those components could have on your experiment.

-any suggestions for dialyzing the ammonium bicarbonate out of the product?

the recombinant Apo Es are shipped in 0.7 M ammonium bicarbonate for stability. If the ammonium bicarbonate concentration cannot be reduced by dilution, it must be reduced by either dialysis or lyophilization. If you choose to dialyze, use standard Spectra PorTM 12,000 Da cutoff tubing. For small quantities of product, use the Dial-a-slideTM system. Step down the ammonium bicarbonate concentration, first to 300 mM and then to the concentration you desire. Be advised that at low ammonium bicarbonate concentrations (<150 mM) Apo E tends to fall out of solution.

What steps should I take when lyophilizing Apo E?

A freeze-drying lyophilizer works best. To remove the high concentration of ammonium bicarbonate (0.7 M), lyophilize for a longer time than you are accustomed. Lyophilized Apo E does not resuspend completely in PBS. For resuspension, use denaturing buffers, particularly those containing urea. Some scientists believe that lyophilization affects the biological activity of Apo E. However, manufacturer tests with lyophilization has had no effect on immunoreactivity (as assayed on Western blots).

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3.0 REFERENCES

LaDu, M.J., et al. (1994) J. Biol. Chem. 269: 23403-23406.

Strittmatter,W.J., et al. (1993) PNAS 88: 8098-8102.

Fleming, L.M., et al. (1996) Exp. Neurol.. 138: 252-260.

Gretch, D., et al. (1991) PNAS 88: 8530-8533.

Laemmli, U.K. (1970) Nature (London) 227: 680.

Spectra Por is a trademark of Spectrum Medical Industries, Inc. Dial-a-slide is a trademark of Pierce Chemical Co.

-copyright by owner(s)


DATA SHEET:  recombinant Human Apo-SAA

Human Apo-SAA is a 104 amino acid polypeptide that ciculates primarily in association with high density lipoproteins (HDL). The level of Apo-SAA, normally 1-5ug/ml in plamsa increases 500-1000 fold within 24 hours of an inflammatory stimulus and under these conditions, is the most abundant HDL apolipoprotein.

Catalog#: RDI-3013

Batch#

Package Size: 50 micrograms $212.00

Supplied: Lyophilized powder with no additives. Endotoxin level is less than 0.1ng per ug Apo- SAA.

Source: E. Coli

Purity: >98% by SDS-Page analysis

Reconstitution: The lyophilized Apo-SAA should be reconstituted in water to a concentration of 100 ng/ul. This solution can be diluted into other buffered solutions or stored at -20 DEG C for future use. For most in-vitro applications, Apo-SAA exerts its biological activity in the range of 5.0 to 50.0 ug/ml.


Storage: The lyophilized powder is stable at room temperature but it is best stored desiccated below 0 Deg C. Reconstituted PF-4 should be stored in working aliquots at -20DEG C. AVOID FREQUENT FREEZE THAW CYCLES.

Precautions: For In vitro research Use Only. Not for use in or on humans or animals or for diagnostics.

It is the responsibility of the user to comply with all local/state and Federal rules in theuse of this product. We are not responsible for any patent infringements that might result with the use of or derivation of this product.


RDI Division of Fitzgerald Industries Intl

34 Junction Square Drive

Concord MA 01742-3049

USA

phone (978) 371-6446 or (800) 370-2222

fax     (978) 371-2266

EMAIL:antibodies@fitzgerald-fii.com

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