Western blot analysis of extracts from Hela cells, either wild type (lane 1) or IDH1 knock-out (lane 2), using IDH1 (RcMab-1) Rat mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). The absence of signal in lane 2 (upper panel) demonstrates specificity of the antibody for IDH1.
Western blot analysis of extracts from various cell lines using IDH1 (RcMab-1) Rat mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). Expression levels of IDH1 among cell lines are consistent with expectations based on publicly available bioinformatic databases.
Western blot analysis of recombinant GST-tagged IDH1 (lane 1) or recombinant GST-tagged IDH2 (lane 2), using IDH1 (RcMab-1) Rat mAb (upper) and GST (91G1) Rabbit mAb #2625 (lower). The absence of signal in lane 2 (upper panel) demonstrates lack of cross-reactivity of the antibody with IDH2.
Immunohistochemical analysis of paraffin-embedded human ductal breast carcinoma using IDH1 (RcMab-1) Rat mAb.
Immunohistochemical analysis of paraffin-embedded human prostate adenocarcinoma using IDH1 (RcMab-1) Rat mAb.
Immunohistochemical analysis of paraffin-embedded human colon adenocarcinoma using IDH1 (RcMab-1) Rat mAb.
Immunohistochemical analysis of paraffin-embedded OV-90 cell pellet (left, high-expressing) or HBP-ALL cell pellet (right, low-expressing) using IDH1 (RcMab-1) Rat mAb.
Immunohistochemical analysis of paraffin-embedded human prostate adenocarcinoma using IDH1 (RcMab-1) Rat mAb (left) or IDH1 Mouse mAb (right). These two antibodies detect independent, unique epitopes on human IDH1. The similar staining patterns obtained with both antibodies help to confirm the specificity of the staining. Note: IDH1 Mouse mAb is weaker than IDH1 (RcMab-1) Rat mAb and, therefore, does not stain all features with the same intensity, but it does help to confirm the specificity of the nuclear staining.
|Immunohistochemistry (Paraffin)||1:50 - 1:200|
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.
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: 340
NOTE: Do not allow slides to dry at any time during this procedure.
For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
posted April 2015
revised March 2016
Protocol Id: 725
IDH1 (RcMab-1) Rat mAb recognizes endogenous levels of total IDH1 protein. This antibody also detects IDH1 when mutated at Arg132 (9).Species Reactivity:
Monoclonal antibody is produced by immunizing animals with recombinant protein corresponding to human IDH1 protein (9,10).
IDH1 is one of three isocitrate dehydrogenases that catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG). These enzymes exist in two distinct subclasses that utilize either NAD or NADP+ respectively, as an electron acceptor (1). IDH1 is the NADP+-dependent isocitrate dehydrogenase found in the cytoplasm and peroxisomes. IDH2 and 3 are mitochondrial enzymes that also function in the Krebs cycle. IDH1 is inactivated by phosphorylation at Ser113 and contains a clasp-like domain wherein both polypeptide chains in the dimer interlock (2,3). IDH1 is expressed in a wide range of species and also in organisms that lack a complete citric acid cycle. Mutations in IDH1 have been reported in glioblastoma (4), acute myeloid leukemia (5,6), and other malignancies (7). IDH1 appears to function as a tumor suppressor that, when mutationally inactivated, contributes to tumorigenesis in part through induction of the HIF-1 pathway (8).
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