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HA-tag control-lysate
Description
The HA-tag control-lysate was generated from transfected HEK293 cells, expressing a HA-tagged protein. The HA-tag corresponds to a peptide of the hemaglutinin protein of the influenza virus. The peptide sequence of the HA-tag (YPYDVPDYA) is not present in any mammalian protein. To generate the HA-tag control-lysate, HEK293 cells were transiently transfected with a mammalian expression vector coding for a murine cytoplasmic protein harbouring a double HA-tag at the carboxy- terminus. A whole cell lysate was prepared from these transiently transfected cells. Besides all other cellular proteins, this whole cell lysate contains the HA-tag protein, which shows an apparent molecular weight of ~45 kDa upon SDS-PAGE.
In addition to the HA-tag control-lysate, a mock-lysate was prepared from HEK293 cells, which were transfected with the empty expression vector.
Product
Whole cell lysate:
50 µl
Cell line:
HEK293 cells
Tagged protein:
Murine cytoplasmic protein with a
carboxy-terminal double HA-tag.
MW:
~45 kDa
Lysis buffer:
60 mM Tris/HCL pH 6.8, 2% SDS,
5% mercaptoethanol,
50 µg/ml bromophenolblue, 10% glycerol
Application:
For SDS-PAGE/Western Blotting
20 µl of the lysates should be applied 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 HA-tag controllysate and the mock-lysate.
HEK293 cells were transiently transfected either
with an expression vector encompassing
the cDNA of a HA-tag protein (HA-tag control-lysate) or with the empty expression vector
(mock-lysate; mock-lys.).
The indicated amounts of the HA-tag control-lysate (30, 20, 10, 5, or 0 µl) or 30 µl of the mock-lysate were separated by SDS-PAGE using
a 10% polyacrylamide-gel and then transfered onto
a PVDF membrane. The membrane was probed
with the monoclonal anti-HA-tag antibody from tag-tools (clone TT7; dilution 1:2,000).
The HA-tag bound primary antibody was
detected by incubation with HRP-coupled
goat-anti-mouse antibody (dilution 1:10,000)
and visualized using chemiluminescence.
The X-ray film was exposed for 10 seconds.
Whereas a specific ~45 kDa band is detected
in the HA-tag control-lysate at all applied volumes
(5 - 30 µl), no binding of the anti-H- tag antibody
to cellular proteins can be detected in the lane,
where 30 µl of the mock-lysate was applied.
Use
The HA-tag control-lysate and the mock-lysate
are for quality control in research applications only.
Not for diagnostic or therapeutic purpose.
