Western blot analysis of extracts from various cell lines and tissues using CRABP1 (D5W9A) Rabbit mAb.
Western blot analysis of extracts from SK-N-AS (positive) and RKO (negative) cells using CRABP1 (D5W9A) Rabbit mAb (upper) and GAPDH (D16H11) XP® Rabbit mAb (lower).
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with Myc/DDK-tagged constructs expressing full-length human CRABP1 (hCRABP1-Myc/DDK; +), full-length human CRABP2 (hCRABP2-Myc/DDK; +); full-length human CRBP1 (hCRBP1-Myc/DDK; +), full-length human CRBP2 (hCRBP2-Myc/DDK; +), and full-length human FABP4 (hFABP4-Myc/DDK; +), using CRABP1 (D5W9A) Rabbit mAb (upper) and DYKDDDDK Tag Antibody #2368 (lower).
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.
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.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
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 October 2016
Protocol Id: 10
CRABP1 (D5W9A) Rabbit mAb recognizes endogenous levels of total CRABP1 protein. This antibody does not cross-react with other intracellular lipid-binding protein family members, CRBP1 and CRBP2.Species Reactivity:
Human, MouseSpecies predicted to react based on 100% sequence homology:
Rat, Chicken, Bovine
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human CRABP1 protein.
Vitamin A gives rise to multiple species of biologically active lipophilic metabolites, known as retinoids, which play a critical role in numerous physiological processes such as vision and embryonic development. Intracellularly, all-trans retinoic acid is bound with high affinity to either cellular retinoic acid-binding protein 1 (CRABP1) or cellular retinoic acid-binding protein 2 (CRABP2), which aids in its solubilization within the aqueous cytosolic compartment. Belonging to the intracellular lipid-binding protein family (iLBP), the human CRABPs are 74% identical at the protein level and each CRABP is highly conserved across multiple species. Research studies have shown that knockout of Crabp1 is not lethal but results in defects in limb development (1), suggesting that CRABP1 plays a role in establishing retinoic acid concentration gradients in the developing limb bud. Although it remains unclear how CRABP1 may regulate the formation of retinoic acid gradients in vivo, research studies have suggested that CRABP1 can enhance the activities of intracellular retinoic acid-metabolizing enzymes, thus blunting cellular responses to retinoic acid (2-4).
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