Antibody data
- Antibody Data
- Antigen structure
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- Validations
- Western blot [1]
- ELISA [1]
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- Product number
- R1172 - Provider product page
- 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)
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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.
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.