Actin expression in protein extracted from tissue
(by L.Kamalian from liverpool.ac.uk)
Thu Apr 2 04:23:57 EST 2009
Hi, I have encountered a few problems after extracting protein from tissue sections. The tissue sections I used were primary tumour sections produced in mouse using sub-coetaneous injection of a lung malignant cell line. The sections were kept in RNA-later solution for 48 hours, then the solution was removed and the sections were transferred to - 80 freezer. To extract protein, the sections were cut into small pieces, then after adding lysing buffer (Cell-lytic M) they were sonicated 3 X 30 seconds. This followed by 30 mins centrifuge, full speed and then the supernatant was removed and the protein concentration was measured by Bradford Assay. All samples showed quite good protein content (3-8 mg/ml). So far so good. Then the problem started: to confirm the protein concentration and also the quality of the samples, 30 ug of each were subjected to polyacrylamide gel electrophoresis, followed by western blot, using mouse anti b-actin mono-clonal antibody as the primary and rabbit anti mouse as the secondary, with the usual dilutions and incubation period. The result is very strange. First of all there are lots of non-specific bands which have never occurred to any of other actin blots before. Secondly, the assumed actin band shows extreme difference in protein loaded. Looking at the gel after transfer, there is not that much difference between the samples. Moreover, the other nonspecific bands are equal!! I am lost. Please help me.
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