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phospho-tyrosine control-lysate  new!

 

Description

The phospho-tyrosine (pTyr) control-lysate was generated from transfected HEK293 cells, expressing the viral protein-tyrosine kinase v-Src. This virus-derived kinase is constitutively active and phosphorylates several cellular proteins, even in serum-starved cells. In contrast, tyrosine phosphorylation of cellular proteins in cells transfected with the empty vector (mock lysate) is barely detectable under these conditions. The cell-lysates were prepared using a lysis buffer, which contains multiple phosphatase- inhibitors to preserve the protein-tyrosine phosphory- lation in the cell lysates. In lysates from v-Src expressing cells (pTyr control lysate) cellular proteins with an apparent molecular weight of ~30, 55, and 75 kDa are the most intense bands detectable by Western Blotting with the anti-pTyr antibody.

Product

Whole cell lysate:
50 µl
Cell line:
HEK293 cells
Overexpressed protein:
v-Src , functioning as a constitutively-
active protein-tyrosine kinase
MW:
multiple pTyr proteins (~30, 55, and 75 kDa)
Lysis buffer:
60 mM Tris/HCL pH 6.8, 2% SDS,
5% mercaptoethanol,
50 µg/ml bromophenolblue, 10% glycerine
Application:
For SDS-PAGE/Western Blotting 20 µl of the lysates should be loaded per lane of a 10% polyacrylamide-gel.

Shipping and storage

Shipping:
The lysate is shipped in cold case.
Storage:
Lysate is stable for 1 week at 4°C. For prolonged storage, the lysate should be stored at -80°C. Aliquot to avoid repeated freezing and thawing. At -80°C, the product is stable for at least 1 year from shipment.

Application in quality control

Western Blotting using the pTyr control lysate and the mock lysate.



Western Blotting using the pTyr control lysate and the mock lysate.
HEK293 cells were transiently transfected either with a eukaryotic expression vector, encoding the cDNA for the protein tyrosine kinase v-Src (pTyr control lysate), or with the empty expression vector (mock lysate; mock lys.). The indicated amounts of the pTyr control-lysate (25, 20, 15, 10, or 5 µl) or 25 µl of the mock lysate, respectively, were separated by SDS-PAGE on a 10% polyacrylamide-gel and then transfered onto a PVDF membrane. The membrane was probed with the monoclonal anti-pTyr antibody from tag-tools (clone TT11; dilution 1:1.000). The bound antibody was detected by incubation with HRP-coupled protein G (dilution 1:10.000) and visualized using chemiluminescence. The X-ray film was exposed for 15 seconds. Multiple tyrosine phosphorylated proteins are visible in 5 - 25 µl of the lysates from v-Src expressing cells (pTyr control lysate), with the most prominent bands at ~30, 55, and 75 kDa. In contrast, barely any tyrosine phosphorylation is detectable in 25 µl of the mock lysate derived from control transfected, serum-starved cells.

Use

The pTyr control lysate and the mock lysate are for quality control in research applications only. They are not intended for diagnostic or therapeutic purposes.


 

pTyr control-lysate