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p-Mek Antibody Staining Protocol for Immunohistochemistry
Description: This antibody detects endogenous levels of MEK1/2 only when phosphorylated at serines 217 and 221. This antibody does not cross-react with related kinases including activated SEK (MKK4), MKK3, or MKK6. It will also react with MEK1/2 singly phosphorylated at Ser217, but not with MEK1/2 singly phosphorylated at Ser221.
Primary Antibody
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Name: phospho-Mek (p-Mek) 1/2 (Ser217/221) Antibody |
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Clone: Rabbit polyclonal |
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Supplier: Cell Signaling Technology |
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Catalog Number: 9121 |
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Dilution: 1:150 using IHC-TekTM Antibody Diluent (Cat# IW-1000 or IW-1001) to reduce background and unspecific staining and serum blocking step is NOT needed. |
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Incubation Time/Temp: 60 min/room temperature |
Antigen Retrieval
| Device: IHC-TekTM Epitope Retrieval Steamer Set (Cat# IW-1102) |
| Buffer/pH value: IHC-TekTM Epitope Retrieval Solution (Cat# IW-1100) |
| Heat/Cool Temperature: 95-100 ºC/room temperature |
| Heat/Cool Time: 20 minutes/20 minutes |
Detection Methods
| Standard Method: ABC Method or LSAB Method |
| Enhanced Method: Polymeric Methods |
Chromogen Substrate
| Reagent: DAB |
| Incubation Time/Temperature: 1-3 minutes/room temperature |
Counterstain
| Reagent: Mayer's Hematoxylin |
| Staining Time: 30 seconds |
Results:
| Staining Pattern: Cytoplasmic/nuclear |
| Images: Search image |
Additional Information:
| Species Reactivity: Mouse |
| Fixation: Formalin fixed paraffin sections |
| Positive Control: Tumor (HT29-xenograph) |
| Negative Control: Omit primary antibody, isotype control, absorption control |
| Blocking: 2-5% normal serum to reduce unspecific background staining; 0.5-3% H2O2 to block endogenous peroxidase activity; avidin/biotin to block endogenous biotin activity if necessary |
References: