WB result of Phospho-IκBα (Ser32/36) Rabbit mAb
Primary antibody: Phospho-IκBα (Ser32/36) Rabbit mAb at 1/1000 dilution
Lane 1: HeLa whole cell lysate 20 µg
Lane 2: HeLa treated with Calyculin A (100ng/ml, 30min) and TNF-α (20ng/ml, 5min) whole cell lysate 20 µg
Lane 3: HeLa whole cell lysate 20 µg (treated with phosphatase)
Lane 4: HeLa treated with Calyculin A (100ng/ml, 30min) and TNF-α (20ng/ml, 5min) whole cell lysate 20 µg (treated with phosphatase)
Secondary antibody: Goat Anti-Rabbit IgG, (H+L), HRP conjugated at 1/10000 dilution
Predicted MW: 36 kDa
Observed MW: 35 kDa
Phospho-IκBα (Ser32/36) Recombinant Rabbit mAb (S-R362)
Phospho-IκBα (Ser32/36) Recombinant Rabbit mAb (S-R362)
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Product Details
Product Details
Product Specification
| Host | Rabbit |
| Antigen | Phospho-IκBα (Ser32/36) |
| Synonyms | NF-kappa-B inhibitor alpha, I-kappa-B-alpha (IkB-alpha; IkappaBalpha), Major histocompatibility complex enhancer-binding protein MAD3, NFKBIA, IKBA, MAD3, NFKBI |
| Location | Cytoplasm, Nucleus |
| Accession | P25963 |
| Clone Number | S-R362 |
| Antibody Type | Recombinant mAb |
| Isotype | IgG |
| Application | WB, IHC-P, ICC, IP |
| Reactivity | Hu, Ms, Rt |
| Purification | Protein A |
| Concentration | 0.5 mg/ml |
| Conjugation | Unconjugated |
| Physical Appearance | Liquid |
| Storage Buffer | PBS, 40% Glycerol, 0.05%BSA, 0.03% Proclin 300 |
| Stability & Storage | 12 months from date of receipt / reconstitution, -20 °C as supplied |
Dilution
| application | dilution | species |
| WB | 1:1000 | |
| IHC-P | 1:500 | |
| ICC | 1:50 | |
| IP | 1:50 |
Background
IκBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha) is one member of a family of cellular proteins that function to inhibit the NF-κB transcription factor. IκBα inhibits NF-κB by masking the nuclear localization signals (NLS) of NF-κB proteins and keeping them sequestered in an inactive state in the cytoplasm. In addition, IκBα blocks the ability of NF-κB transcription factors to bind to DNA, which is required for NF-κB's proper functioning.
Picture
Picture
Western Blot
IP
Phospho-IκBα (Ser32/36) Rabbit mAb at 1/50 dilution (1 µg) immunoprecipitating Phospho-IκBα (Ser32/36) in 0.4 mg HeLa treated with Calyculin A (100ng/ml, 30min) and TNF-α (20ng/ml, 5min) whole cell lysate.
Western blot was performed on the immunoprecipitate using Phospho-IκBα (Ser32/36) Rabbit mAb at 1/1000 dilution.
Secondary antibody (HRP) for IP was used at 1/400 dilution.
Lane 1: HeLa treated with Calyculin A (100ng/ml, 30min) and TNF-α (20ng/ml, 5min) whole cell lysate 20 µg (Input)
Lane 2: Phospho-IκBα (Ser32/36) Rabbit mAb IP in HeLa treated with Calyculin A (100ng/ml, 30min) and TNF-α (20ng/ml, 5min) whole cell lysate
Lane 3: Rabbit monoclonal IgG IP in HeLa treated with Calyculin A (100ng/ml, 30min) and TNF-α (20ng/ml, 5min) whole cell lysate
Predicted MW: 36 kDa
Observed MW: 35 kDa
Immunohistochemistry
IHC shows positive staining in paraffin-embedded human breast cancer (Left) and negative staining in phosphatase treated (37 °C, 2h) section (Right). Anti-Phospho-IκBα (Ser32/36) antibody was used at 1/500 dilution, followed by a HRP Polymer for Mouse & Rabbit IgG (ready to use). Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris/EDTA buffer pH9.0 was performed before commencing with IHC staining protocol.
IHC shows positive staining in paraffin-embedded human tonsil (Left) and negative staining in phosphatase treated (37 °C, 2h) section (Right). Anti-Phospho-IκBα (Ser32/36) antibody was used at 1/500 dilution, followed by a HRP Polymer for Mouse & Rabbit IgG (ready to use). Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris/EDTA buffer pH9.0 was performed before commencing with IHC staining protocol.
IHC shows positive staining in paraffin-embedded mouse spleen (Left) and negative staining in phosphatase treated (37 °C, 2h) section (Right). Anti-Phospho-IκBα (Ser32/36) antibody was used at 1/500 dilution, followed by a HRP Polymer for Mouse & Rabbit IgG (ready to use). Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris/EDTA buffer pH9.0 was performed before commencing with IHC staining protocol.
IHC shows positive staining in paraffin-embedded rat spleen (Left) and negative staining in phosphatase treated (37 °C, 2h) section (Right). Anti-Phospho-IκBα (Ser32/36) antibody was used at 1/500 dilution, followed by a HRP Polymer for Mouse & Rabbit IgG (ready to use). Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris/EDTA buffer pH9.0 was performed before commencing with IHC staining protocol.
Immunocytochemistry
ICC analysis of HeLa cells treated with TNF-α (20ng/ml, 5min) (top panel) and HeLa cells untreated with TNF-α (20ng/ml, 5min) (below panel). Anti-Phospho-IκBα (Ser32/36) antibody was used at 1/50 dilution (Green) and incubated overnight at 4°C. Goat polyclonal Antibody to Rabbit IgG - H&L (Alexa Fluor® 488) was used as secondary antibody at 1/1000 dilution. The cells were fixed with 4% PFA and permeabilized with 0.1% PBS-Triton X-100. Nuclei were counterstained with DAPI (Blue). Counterstain with tubulin (Red).
