Western blot analysis of extracts from HeLa cells, transfected with a construct expressing full-length human ApoE2 (hApoE2; +), ApoE3 (hApoE3; +), or ApoE4 (hApoE4; +), using ApoE4 (4E4) Mouse mAb (upper) or a pan ApoE antibody (lower).Learn more about how we get our images.
Western blot analysis of human serum using ApoE4 (4E4) Mouse mAb.Learn more about how we get our images.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised June 2016
Protocol Id: 262
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
ApoE4 (4E4) Mouse mAb recognizes endogenous levels of total ApoE4 protein. This antibody does not cross-react with ApoE2 or ApoE3.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Arg130 of human ApoE4 protein.
Apolipoproteins are plasma lipoproteins that function as transporters of lipids and cholesterol in the circulatory system. Chylomicrons are a fundamental class of apolipoproteins containing very low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL) (1,2).
Human ApoE has three isoforms: ApoE2, ApoE3, and ApoE4. These three isoforms differ in the combination of cysteine and arginine residues located at positions 130 and 176. The ApoE4 isoform contains arginine at both locations. Research studies have linked ApoE4 function to neuronal plasticity, synaptogenesis, and neurodegenerative diseases (3). ApoE4 is produced in the liver and brain, although it is widely expressed in other tissues, such as the lung, spleen, and ovary. Investigators have established the ApoE4 allele as a genetic risk factor for Alzheimer’s disease (AD), accounting for 50-60% of the genetic variation in the disease (4). Research studies indicate that patients expressing ApoE4 have a reduced capacity for synaptic plasticity, an earlier age of onset of AD, and an increase in amyloid-beta (Aβ) deposition. The increase in Aβ suggests a role for ApoE4 in the impairment of amyloid clearance (5).
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