Anti-ASK1/MAP3K5 Antibody Picoband™

Product Name

Anti-ASK1/MAP3K5 Antibody Picoband™

View all ASK1 Antibodies

SKU/Catalog Number

A00929-3

Size

100 μg/vial

Form

Lyophilized

Description

Boster Bio Anti-ASK1/MAP3K5 Antibody Picoband™ catalog # A00929-3. Tested in ELISA, Flow Cytometry, IF, IHC, ICC, WB applications. This antibody reacts with Human, Mouse, Rat.

Storage & Handling

At -20°C for one year from date of receipt. After reconstitution, at 4°C for one month. It can also be aliquotted and stored frozen at -20°C for six months. Avoid repeated freezing and thawing.

Cite This Product

Anti-ASK1/MAP3K5 Antibody Picoband™ (Boster Biological Technology, Pleasanton CA, USA, Catalog # A00929-3)

Host

Rabbit

Contents

Each vial contains 4 mg Trehalose, 0.9 mg NaCl, 0.2 mg Na2HPO4.

Clonality

Polyclonal

Isotype

Rabbit IgG

Immunogen

E.coli-derived human ASK1/MAP3K5 recombinant protein (Position: A178-K1112).

*Blocking peptide can be purchased. Costs vary based on immunogen length. Contact us for pricing.

Cross-reactivity

No cross-reactivity with other proteins.

Reactive Species

A00929-3 is reactive to MAP3K5 in Human, Mouse, Rat

Applications

A00929-3 is guaranteed for ELISA, Flow Cytometry, IF, IHC, ICC, WB Boster Guarantee

Observed Molecular Weight

155 kDa

Calculated molecular weight

154.537kDa

Background of ASK1

Apoptosis signal-regulating kinase 1 (ASK1) also known as mitogen-activated protein kinase 5 (MAP3K5) is a member of MAP kinase family and as such a part of mitogen-activated protein kinase pathway. Mitogen-activated protein kinase (MAPK) signaling cascades include MAPK or extracellular signal-regulated kinase (ERK), MAPK kinase (MKK or MEK), and MAPK kinase kinase (MAPKKK or MEKK). MAPKK kinase/MEKK phosphorylates and activates its downstream protein kinase, MAPK kinase/MEK, which in turn activates MAPK. The kinases of these signaling cascades are highly conserved, and homologs exist in yeast, Drosophila, and mammalian cells. MAPKKK5 contains 1,374 amino acids with all 11 kinase subdomains. Northern blot analysis shows that MAPKKK5 transcript is abundantly expressed in human heart and pancreas. The MAPKKK5 protein phosphorylates and activates MKK4 (aliases SERK1, MAPKK4) in vitro, and activates c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) during transient expression in COS and 293 cells; MAPKKK5 does not activate MAPK/ERK.

Antibody Validation

Boster validates all antibodies on WB, IHC, ICC, Immunofluorescence, and ELISA with known positive control and negative samples to ensure specificity and high affinity, including thorough antibody incubations.

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Reconsitution

Adding 0.2 ml of distilled water will yield a concentration of 500 μg/ml.

Assay Dilutions Recommendation

The recommendations below provide a starting point for assay optimization. The actual working concentration varies and should be decided by the user.

Western blot, 0.25-0.5 μg/ml, Human, Mouse, Rat
Immunohistochemistry(Paraffin-embedded Section), 2-5 μg/ml, Human
Immunocytochemistry/Immunofluorescence, 5 μg/ml, Human
Flow Cytometry, 1-3 μg/1x10⁶ cells, Human
Direct ELISA, 0.1-0.5 μg/ml, Human

Validation Images & Assay Conditions

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Figure 1. Western blot analysis of ASK1/MAP3K5 using anti-ASK1/MAP3K5 antibody (A00929-3).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: human RT4 whole cell lysates,
Lane 2: human HEL whole cell lysates,
Lane 3: human HL-60 whole cell lysates,
Lane 4: human THP-1 whole cell lysates,
Lane 5: rat liver tissue lysates,
Lane 6: mouse liver tissue lysates,
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-ASK1/MAP3K5 antigen affinity purified polyclonal antibody (Catalog # A00929-3) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for ASK1/MAP3K5 at approximately 155 kDa. The expected band size for ASK1/MAP3K5 is at 155 kDa.

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Figure 2. IHC analysis of ASK1/MAP3K5 using anti-ASK1/MAP3K5 antibody (A00929-3).
ASK1/MAP3K5 was detected in a paraffin-embedded section of human lung cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-ASK1/MAP3K5 Antibody (A00929-3) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1022) with DAB as the chromogen.

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Figure 3. IHC analysis of ASK1/MAP3K5 using anti-ASK1/MAP3K5 antibody (A00929-3).
ASK1/MAP3K5 was detected in a paraffin-embedded section of human spleen tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-ASK1/MAP3K5 Antibody (A00929-3) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1022) with DAB as the chromogen.

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Figure 4. IF analysis of ASK1/MAP3K5 using anti-ASK1/MAP3K5 antibody (A00929-3).
ASK1/MAP3K5 was detected in an immunocytochemical section of MCF-7 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 μg/mL rabbit anti-ASK1/MAP3K5 Antibody (A00929-3) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

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Figure 5. Flow Cytometry analysis of Raji cells using anti-ASK1/MAP3K5 antibody (A00929-3).
Overlay histogram showing Raji cells stained with A00929-3 (Blue line). The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-ASK1/MAP3K5 Antibody (A00929-3, 1 μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10 μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1 μg/1x10⁶) used under the same conditions. Unlabelled sample (Red line) was also used as a control.

Gene/Protein Information For MAP3K5 (Source: Uniprot.org, NCBI)

Alternative Names

Apoptosis signal-regulating kinase 1; ASK1; ASK-1; ASK1MEKK 5; EC 2.7.11; MAP/ERK kinase kinase 5; MAP3K5; MAPK/ERK kinase kinase 5; MAPKKK5EC 2.7.11.25; MEK kinase 5; MEKK5; MEKK5apoptosis signal regulating kinase 1; mitogen-activated protein kinase kinase kinase 5 MAP3K5 ASK1, MAPKKK5, MEKK5 mitogen-activated protein kinase kinase kinase 5 mitogen-activated protein kinase kinase kinase 5|ASK-1|MAP/ERK kinase kinase 5|MAPK/ERK kinase kinase 5|MEK kinase 5|MEKK 5|apoptosis signal-regulating kinase 1

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