Method for simultaneously turning two films and marking protein TRX and procaspase12
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A technology of labeling protein and labeling, applied in the field of molecular biology
Inactive Publication Date: 2009-08-05
KUNMING UNIV OF SCI & TECH
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Embodiment 1
[0024] One gel was transferred to two PVDF membranes, and two antibodies were labeled at the same time:
[0025] (1) Extraction of total cell protein: After stimulating PC12 cells with drugs for 24 hours, discard the medium, add about 2ml of PBS to wash the remaining medium on the cell surface, and discard the washing solution. Add 0.25% trypsin to digest for about 5min to stop the digestion, centrifuge at 1500rpm for 5min, discard the supernatant, add 1ml PBS to suspend cells, centrifuge at 1500rpm for 5min at 4°C, discard the supernatant, add 100μl cell lysate, lyse on ice for about 1h, 15000rpm 4°C Centrifuge for 15 minutes, transfer the supernatant (total cell protein) to a new Dorf tube, and store at -80°C.
[0026] (2) Protein quantification: operate according to the operation instructions of the BCA protein quantification kit, and determine the concentration of the sample.
[0027] (3) Electrophoresis: The extracted total cell protein was subjected to denatured polyacr...
Embodiment 2
[0034] Accuracy and stability of the method for detecting embodiment:
[0035] The accuracy of the present invention was tested by labeling with two different antibodies using the traditional stripping method.
[0036] The specific method is as follows:
[0037] (1) Add 10ml stripping solution (0.27ml of β-mercaptoethanol, 2ml of 10% SDS, 625μl of 1.0M Tris 6.8) to the PVDF membrane labeled with TRX antibody that has been developed on X-ray film, incubate at 60°C for 30min, wash with TBPS Wash three times for 10 minutes, and then put in 5% skimmed milk to block overnight at 4°C.
[0038] (2) The membrane was taken out from the skim milk, washed three times with TPBS for 10 min, labeled with procaspase 12 primary antibody, and incubated for 70 min.
[0039] (3) After the primary antibody is labeled, wash with TPBS three times for 5 minutes, label the secondary antibody, and incubate for 70 minutes.
[0040] (4) After the secondary antibody labeling is completed, wash with TP...
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Abstract
The invention relates to a method for simultaneously rotating two films and marking two proteins, namely TRX and procaspase12. The method includes the steps of total protein extraction of cells, protein quantifying, gel electrophoresis, protein blotting, simultaneously marking of antibodies, X-ray film developing, and the like. The invention aims at building up a method which is characterized in that two PVDF films are used for simultaneously marking and detecting the two target proteins by using different antibodies after SDS-PAGE and protein blotting are carried out on the total protein in cells, meanwhile, the method is compared with the traditional method which has the steps of stripping films and then marking antibodies, and the accuracy, the reproducibility, the stability, and the like of the method are evaluated. The invention is simple and easy to carry out, can well represent the test result, and provides a method which is simpler and easier and saves time and samples, thereby having actual application value.
Description
technical field [0001] The invention belongs to the field of molecular biology and relates to a method for simultaneously transfecting two membranes and marking protein TRX and procaspase 12, so as to detect the changes of the two target proteins. Background technique [0002] Western Blot is a method for detecting a certain protein in complex samples based on the specific binding of antigen and antibody. [0003] Western blot is a new immunobiochemical technique developed on the basis of gel electrophoresis and solid-phase immunoassay technology, mainly including gel electrophoresis, western blotting (membrane transfer) and immunological detection. [0004] Gel electrophoresis is to divide the protein in the sample to be tested into bands in the gel according to the molecular weight. Western blotting refers to the transfer of protein samples separated by gel electrophoresis to a solid phase support (such as nitrocellulose membrane or PVDF membrane). Its biological activit...
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