R1172

antibody from Acris Antibodies GmbH

Targeting: MAP3K5 ASK1, MAPKKK5, MEKK5

Western blot (Supportive data in Antibodypedia)

ELISA (Supportive data in Antibodypedia)

Antibody Data

Product number

R1172

Provider

Acris Antibodies GmbH

Proper citation

Acris Antibodies GmbH Cat#R1172, RRID:AB_973032

Product name

anti MEKK5 / ASK1 pSer83

Antibody type

Polyclonal

Antigen

This purified antibody was prepared from rabbit serum after repeated immunizations with a KLH conjugated peptide corresponding to amino acids 76-87 of human ASK-1 protein.

Reactivity

Human

Host

Rabbit

Vial size

0.2 mg

Concentration

2.0 mg/ml (by UV absorbance at 280 nm)

Western blot

Supportive validation

Submitted by

Acris Antibodies GmbH (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 transienttransfection, 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 24h post-transfection in 200 µL of 1x SDS-sample buffer, heatedat 96°C for 5', and vortexed for 30 sec. Samples (10 µL each)were separated on a 12% SDS-PAGE gel and transferred toPVDF (Millipore) followed by blocking for 45' using TTBSsupplemented with 5% non-fat dry milk. All incubations wereperformed at room temperature.In panel a) all samples were incubated with 10 µg/mL mouseanti-HA antibody for 45'. After 5X washes with TTBS, reactionwith ALP rabbit anti-mouse IgG at 200 ng/mL proceeded for45' following again by washing as before. The blot wasdeveloped using BCIP/NBT. This blot demonstrates bothrecombinant transfections were successfully over-expressedin the Cos-7 cells.In panel b) all samples were incubated with a 1:1,000 dilution of ASK1 antibody for 45'. The antibodywas pre-incubated with non-phospho peptide prior tomembrane incubation. After 5X washes with TTBS, reactionwith HRP goat anti-rabbit IgG at 10 ng/mL proceeded for 45'following again by washing as before. The membrane wasprocessed as before. Lane 2 shows strong specific stainingof 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 preincubatedwith phospho peptide prior to membrane incubation. No staining is observed after phospho peptide blocking occurs.

ELISA

Supportive validation

Submitted by

Acris Antibodies GmbH (provider)

Main image

Experimental details

Figure 2. ELISA results of purified polyclonalanti-pS83 ASK-1 (aa 76-87) antibody tested against BSA conjugates of non-phospho andphospho forms of immunizing peptide. Each well was coated with 0.1 mg of conjugate. Thestarting dilution of antibody was 1:1,000 and each point on the X-axis represents a 2-folddilution. HRP conjugated Gt-a-Rabbit IgG H&L and TMB substrate were used for detection.