Method for detecting protein in oocytes
An oocyte and detection method technology, applied in the field of protein detection, can solve the problems of limited sample loading, inconvenient detection, scarcity of embryos, etc., and achieve the effect of improving efficiency and efficiency
Active Publication Date: 2017-12-01
ANHUI AGRICULTURAL UNIVERSITY
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[0002] Since Western blot was proposed by Harry Towbin in 1979, it has become a routine method for detecting protein expression levels, and its application in tissue samples and cell samples has become more and more mature. However, when detecting oocytes and embryo samples However, it is limited due to the scarcity of embryos and the limitation of the amount of samples. When it is necessary to detect proteins expressed in low abundance, sometimes hundreds or thousands of embryos are required for one experiment. The detection has brought a lot of inconvenience
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[0030] The technical solutions of the present invention will be described in detail below in conjunction with specific embodiments.
[0031] A method for detecting protein in oocytes, comprising the following steps
[0032] S1, protein sample extraction, prepare 10μl lysate in 0.5ml ep tube A pre-cooled at 4℃, including 9μl WB and IP cell lysate and 1μl PMSF (1mmol / L), take the prepared lysate and add it to ep tube In B, add 15 glass beads with a diameter of 425-600um pre-cooled at 4°C to centrifuge tube B, wash the embryos in DPBS pre-cooled at 4°C for 3 times, transfer them to centrifuge tube B, and vortex The instrument oscillates at 2500rpm for 30s and repeats 3 times. The oscillating is intermittent, with an interval of 30s. The third oscillating is instantaneous centrifugation and then oscillating, then centrifuging. The centrifuging conditions are 4°C, 12000rpm, 4min, and the supernatant is taken Add liquid into centrifuge tube C, add SDS-PAGE protein sample buffer to ...
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Abstract
The invention belongs to the field of protein detection, and in particular relates to a method for detecting protein in oocytes. The method comprises the following steps: S1, extracting a protein sample; S2, performing SDS-PAGE gel electrophoresis flow; S3, transferring a membrane; S4, closing; S5, incubating a primary antibody; S6, incubating a secondary antibody; and S7, developing. A method for completely extracting the protein by adding glass beads in the extraction process of the protein sample and a self-closing bag in the developing process are cleverly applied. Compared with the existing technical method, the method has the advantages that creative invention and improvement are implemented in the treatment mode of the oocyte sample and the optimization aspect of the developing link, so the protein detection efficiency is obviously improved. According to the method and through the scheme that low-abundance protein can be detected by a small amount of test materials, a stripe can be detected by only one oocyte during detection of internal reference protein, a clear stripe can be detected by about 40 oocytes during detection of protein with low expressive abundance, and the efficiency is obviously improved compared with that in the prior art which needs no less than 200 oocytes.
Description
technical field [0001] The invention belongs to the field of protein detection, and in particular relates to a method for detecting protein in oocytes. Background technique [0002] Since Western blot was proposed by Harry Towbin in 1979, it has become a routine method for detecting protein expression levels, and its application in tissue samples and cell samples has become more and more mature. However, when detecting oocytes and embryo samples However, it is limited due to the scarcity of embryos and the limitation of the amount of samples. When it is necessary to detect proteins expressed in low abundance, sometimes hundreds or thousands of embryos are required for one experiment. The detection has brought a lot of inconvenience. Contents of the invention [0003] The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art and provide a detection method for proteins in oocytes, especially for proteins with low expression...
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Login to View More IPC IPC(8): G01N33/68G01N33/561
CPCG01N33/561G01N33/6803
Inventor张运海李辉曹祖兵丁彪罗磊洪仁芸高迪刘成雪尚慧许腾腾
OwnerANHUI AGRICULTURAL UNIVERSITY