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

Name: phospho-Mek (p-Mek) 1/2 (Ser217/221) Antibody

Clone: Rabbit polyclonal

Supplier: Cell Signaling Technology

Catalog Number: 9121

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.

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

Reagent: Mayer's Hematoxylin
Staining Time: 30 seconds

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