MDH1 Antibody (JE35-03)

Western blot shows lysates of HAP1 parental cell line and MDH1 knockout HAP1 cell line (KO). Nitrocellulose membrane was probed with MDH1 Antibody (JE35-03) (Catalog # NBP3-32582) followed by HRP-conjugated secondary antibody. A specific band was detected for MDH1 at approximately 36.4 kDa (as indicated) in the parental HAP1 cell line, but is not detectable in knockout HAP1 cell line. Primary antibody dilution used: 1/1000. The Ponceau stained transfer of the blot is shown. This experiment was conducted under reducing conditions. Image, protocol, and testing courtesy of YCharOS Inc. See ycharos.com for additional details.

HAP1 cell line lysates were prepared and immunoprecipitation was performed using 2.0 μg of MDH1 Antibody (JE35-03) (Catalog # NBP3-32582) pre-coupled to Dynabeads Protein A. Immunoprecipitated MDH1 was detected in Western Blot with a rabbit MDH1 antibody used at 1/10000. The Ponceau stained transfer of the blot is shown. SM=4% starting material; UB=4% unbound fraction; IP=immunoprecipitate; HC=antibody heavy chain. Image, protocol and testing courtesy of YCharOS Inc. (ycharos.com).

HAP1 parental cell line WT and MDH1 HAP1 KO cells were labelled with a green or a far-red fluorescent dye, respectively. Cells were stained with MDH1 Antibody (JE35-03) (Catalog # NBP3-32582) followed by incubation with an Alexa-fluor 555 conjugated secondary antibody (upper panel). DAPI-only counterstained cells shown on a lower panel. Acquisition of the blue (nucleus-DAPI), green (identification of WT cells), red (antibody staining) and far-red (identification of KO cells) channels was performed. Representative images of the blue and red (grayscale) channels are shown. WT and KO cells are outlined with green and magenta dashed line, respectively. Primary antibody dilution used: 1/500. Image, protocol and testing courtesy of YCharOS Inc. (ycharos.com).

Western Blot: MDH1 Antibody (JE35-03) [NBP3-32582] - Western blot analysis of MDH1 on different lysates with Rabbit anti-MDH1 antibody (NBP3-32582) at 1/1,000 dilution. Lane 1: Jurkat cell lysate (20 ug/Lane) Lane 2: HepG2 cell lysate (20 ug/Lane) Lane 3: U-937 cell lysate (20 ug/Lane) Lane 4: HL-60 cell lysate (20 ug/Lane) Lane 5: NIH/3T3 cell lysate (20 ug/Lane) Lane 6: Rat kidney tissue lysate (40 ug/Lane) Predicted band size: 36 kDa Observed band size: 35 kDa Exposure time: 6 seconds; ECL; 4-20% SDS-PAGE gel. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (NBP3-32582) at 1/1,000 dilution was used in 5% NFDM/TBST at 4 overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1/50,000 dilution was used for 1 hour at room temperature.

Immunocytochemistry/ Immunofluorescence: MDH1 Antibody (JE35-03) [NBP3-32582] - Immunocytochemistry analysis of U-937 cells labeling MDH1 with Rabbit anti-MDH1 antibody (NBP3-32582) at 1/100 dilution. Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-MDH1 antibody (NBP3-32582) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI. Beta tubulin (red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594) was used as the secondary antibody at 1/1,000 dilution.

Flow Cytometry: MDH1 Antibody (JE35-03) [NBP3-32582] - Flow cytometric analysis of U-937 cells labeling MDH1. Cells were fixed and permeabilized. Then stained with the primary antibody (NBP3-32582, 1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

• Reactivity: Hu, Mu, Rt
• Applications: WB, Flow, ICC/IF, IP, KO
• Clone: JE35-03
• Clonality: Monoclonal
• Host: Rabbit
• Conjugate: Unconjugated
• Concentration: 1 mg/ml

MDH1 Antibody (JE35-03) Summary

• Description: Novus Biologicals Knockout (KO) Validated Rabbit MDH1 Antibody (JE35-03) (NBP3-32582) is a recombinant monoclonal antibody validated for use in WB, Flow, ICC/IF and IP. All Novus Biologicals antibodies are covered by our 100% guarantee.
• Additional Information: Recombinant Monoclonal Antibody
• Immunogen: Synthetic peptide. (Uniprot: P40925)
• Localization: Cytoplasm, cytosol.
• Source: HEK293
• Isotype: IgG
• Clonality: Monoclonal
• Host: Rabbit
• Gene: MDH1
• Purity: Protein A purified

Applications/Dilutions

• Dilutions:
  • Flow Cytometry 1:1000
  • Immunocytochemistry/ Immunofluorescence 1:100
  • Immunoprecipitation Validated for Immunoprecipitation from YCharOS Inc. (YCharOS.com)
  • Knockout Validated Knockout Validated from YCharOS Inc. (YCharOS.com)
  • Western Blot 1:1000
• Theoretical MW: 36 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Packaging, Storage & Formulations

• Storage: Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.
• Buffer: 1*TBS (pH7.4), 0.05% BSA and 40% Glycerol
• Preservative: 0.05% Sodium Azide
• Concentration: 1 mg/ml
• Purity: Protein A purified

Alternate Names for MDH1 Antibody (JE35-03)

• aromatic alpha-keto acid reductase
• cytoplasmic
• Cytosolic malate dehydrogenase
• DEE88
• Diiodophenylpyruvate reductase
• EC 1.1.1.37
• EC 1.1.1.96
• EIEE88
• epididymis secretory protein Li 32
• HEL-S-32
• KAR
• malate dehydrogenase 1, NAD (soluble)
• Malate dehydrogenase, cytoplasmic
• malate dehydrogenase, cytoplasmic; malate dehydrogenase, peroxisomal
• MDH1
• MDHA
• MDH-s
• MGC:1375
• MOR2
• soluble malate dehydrogenase

Background

Malate dehydrogenase catalyzes the reversible oxidation of malate to oxaloacetate, utilizing the NAD/NADH cofactor system in the citric acid cycle. The protein encoded by this gene is localized to the cytoplasm and may play pivotal roles in the malate-aspartate shuttle that operates in the metabolic coordination between cytosol and mitochondria. (provided by RefSeq)

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Bioinformatics

• Gene Symbol: MDH1