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SV40 Antibody Staining Protocol for Immunohistochemistry
Description: Simian virus 40 is a small DNA virus encoded by 5.2 kb of double-stranded DNA. SV40 large T antigen (T-ag) is a multifunctional ~85 kD phosphoprotein, which is the sole viral protein required for SV40 replication. All other factors are provided by the infected host cell. In addition to its role in SV40 DNA replication, T-ag also causes transformation of susceptible cell lines. Studies of various mutant T-ag proteins have shown that the replication and transformation fractions of T-ag can be separated. The multifunctional nature of this protein has resulted in its use as a model system in a wide variety of disciplines. SV40 T-ag exercises negative regulation on the transcription of SV40 early mRNA by feedback inhibition and exerts positive regulation on transcription from the late promoter. In addition to transcriptional regulation, T-ag is involved in viral DNA replication. Specific biochemical functions required for DNA synthesis that are inherent to the T-ag include high-affinity binding to sites within the viral origin of DNA synthesis, and ATPase and helicase activities. Other functions attributed to T-ag include cellular transformation, induction of cellular DNA synthesis, induction of rRNA synthesis and provision of a host-range function for viral replication. However, all functions of T-ag are influenced by a wide range of post-translational modifications including phosphorylation, glycosylation, acetylation, acylation and adenylation. T-ag exists in monomeric as well as polymeric forms and associates with the tumor suppressor proteins p53 and Rb (retinoblastoma protein). Most of T-ag is transported to the nucleus, while a small fraction is localized at the cell surface.
Primary Antibody
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Name: SV40 Large T Antigen Antibody |
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Clone: PAb 101, Mouse monoclonal |
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Supplier: Pharmingen |
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Catalog Number: 554149 |
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Dilution: 1:100 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: Nuclear |
| Images: Search image |
Additional Information:
| Species Reactivity: Human |
| Fixation: Formalin fixed paraffin sections |
| Positive Control: COS-7 (ATCC CRL 1651), mammary carcinoma |
| 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: