R1172

antibody from OriGene

Targeting: MAP3K5 ASK1, MAPKKK5, MEKK5

Provider product page for R1172

Western blot

ELISA

Antibody data

Antibody Data
Antigen structure
References [0]
Comments [0]
Validations
Western blot [1]
ELISA [1]

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Product number: R1172 - Provider product page
Provider: OriGene
Product name: ASK1 (MAP3K5) pSer83 rabbit polyclonal antibody, Purified
Antibody type: Polyclonal
Description: ASK1 (MAP3K5) pSer83 rabbit polyclonal antibody, Purified
Host: Rabbit
Conjugate: Unconjugated
Epitope: MAP3K5
Antibody clone number: NULL
Vial size: 200 µg
Concentration: 2.0 mg/ml (by UV absorbance at 280 nm)

MAP3K5 protein structure - R1172 shown in red.

Supportive validation

Submitted by: OriGene (provider)
Main image
Experimental details: Figure 1. Immunoblot of anti-pS83 ASK1 antibodies shows specificity for phosphorylated human ASK1. Anti-pS83 (aa 76-87) antibody, generated by immunization with phospho peptide coupled to KLH, was tested by immunoblot against lysates of Cos-7 cells after transient transfection, separately, with 1) vector only, 2) recombinant HA-ASK1, and 3) recombinant human HA-ASK1 where S83 was substituted with an alanine residue. Cells were lysed 24 h post-transfection in 200 ?L of 1x SDS-sample buffer, heated at 96°C for 5', and vortexed for 30 sec. Samples (10 ?L each) were separated on a 12% SDS-PAGE gel and transferred to PVDF (Millipore) followed by blocking for 45' using TTBS supplemented with 5% non-fat dry milk. All incubations were performed at room temperature. In panel a) all samples were incubated with 10 ug/mL mouse anti-HA antibody for 45'. After 5X washes with TTBS, reaction with ALP rabbit anti-mouse IgG at 200 ng/mL proceeded for 45' following again by washing as before. The blot was developed using BCIP/NBT. This blot demonstrates both recombinant transfections were successfully over-expressed in the Cos-7 cells. In panel b) all samples were incubated with a 1:1,000 dilution of ASK1 antibody for 45'. The antibody was pre-incubated with non-phospho peptide prior to membrane incubation. After 5X washes with TTBS, reaction with HRP goat anti-rabbit IgG at 10 ng/mL proceeded for 45' following again by washing as before. The membrane was processed as before. Lane 2 shows strong specific staining of ASK1. Lane 3, where S83 was replaced with alanine, shows greatly diminished staining. In panel c) all samples were incubated with a 1:1,000 dilution of ASK1 antibody as before except the antibody was preincubated with phospho peptide prior to membrane incubation. No staining is observed after phospho peptide blocking occurs.
Validation comment: WB