Cumbers:protocols/Invitrogen NuPage gels: Difference between revisions
John Cumbers (talk | contribs) (New page: =Phosphorylated epitope?= *http://www.sigmaaldrich.com/Brands/Sigma_Genosys/Custom_Antisera/Key_Resources/Antisera_FAQ/Western_Blot.html If you are using an antibody that is specific to ...) |
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=Phosphorylated epitope?= | =Phosphorylated epitope?= | ||
*http://www.sigmaaldrich.com/Brands/Sigma_Genosys/Custom_Antisera/Key_Resources/Antisera_FAQ/Western_Blot.html | *http://www.sigmaaldrich.com/Brands/Sigma_Genosys/Custom_Antisera/Key_Resources/Antisera_FAQ/Western_Blot.html | ||
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=From Brodsky Genome Biology Paper= | =From Brodsky Genome Biology Paper= | ||
8WG16 recognize the hypophosphorylated (PolIIa) | *8WG16 recognize the hypophosphorylated (PolIIa) | ||
4H8, a phosphorylation-independent state of the CTD of polymerase II (PolII) | *4H8, a phosphorylation-independent state of the CTD of polymerase II (PolII) | ||
Thus, the 4H8 antibody is recognizing the total polymerase II population. | *Thus, the 4H8 antibody is recognizing the total polymerase II population. | ||
=4H8= | |||
http://www.abcam.com/index.html?datasheet=5408 | |||
RNA polymerase II carboxy-terminal domain (CTD) interacts with a large multisubunit complex that contains TATA-binding protein (TBP) and is an integral part of the transcription initiation complex. Phosphorylation of RNA polymerase II's largest subunit C-terminal domain (CTD) is a key event during mRNA metabolism. Numerous enzymes, including cell cycle-dependent kinases and TFIIF-dependent phosphatases target the CTD. | |||
*ChIP: Use 1-4µg per 25µg of chromatin. | |||
Latest revision as of 08:10, 10 May 2007
Phosphorylated epitope?
If you are using an antibody that is specific to a phosphorylated epitope, DO NOT use milk to block. Kinases in the milk will nonspecifically phosphorylate proteins in your sample. Use BSA as a blocking agent. It is important to block at least overnight. BSA is not as good of a blocking agent and you will see more background.
From Brodsky Genome Biology Paper
- 8WG16 recognize the hypophosphorylated (PolIIa)
- 4H8, a phosphorylation-independent state of the CTD of polymerase II (PolII)
- Thus, the 4H8 antibody is recognizing the total polymerase II population.
4H8
http://www.abcam.com/index.html?datasheet=5408 RNA polymerase II carboxy-terminal domain (CTD) interacts with a large multisubunit complex that contains TATA-binding protein (TBP) and is an integral part of the transcription initiation complex. Phosphorylation of RNA polymerase II's largest subunit C-terminal domain (CTD) is a key event during mRNA metabolism. Numerous enzymes, including cell cycle-dependent kinases and TFIIF-dependent phosphatases target the CTD.
- ChIP: Use 1-4µg per 25µg of chromatin.
