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A method for removing non-specific hybridization in Western blot experiments
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An immunoblotting, non-specific technique, applied in the field of biological research, can solve the problems of deep hybridization background, RuBisCo large subunit band contamination, and uncertainty of experimental results
Active Publication Date: 2014-10-15
BEIJING PROTEIN INNOVATION
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Because of the high abundance expression of RuBisCo in plants, especially in leaves, it is possible to produce antibodies and plant endogenous RuBisCo large subunits while conducting protein expression research on target genes, especially research on disease resistance and stress resistance. The non-specific hybridization caused the uncertainty of the experimental results, mainly manifested as a deep hybridization background and obvious RuBisCo large subunit band contamination
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Embodiment 1
[0014] Cloning of embodiment 1, RuBisCo large subunit gene
[0015] Amplification primers were designed according to the sequence of the RuBisCo large subunit (Ribulose-1,5bisphosphate carboxylase / oxygenase large subunit N-methyltransferase, chloroplastic) gene (SEQ ID NO: 1) published in the TIGR database, and the rice cDNA library was used as a template for amplification. increase( figure 1 ), recovery, enzyme digestion and connection to the pET-30a expression vector, and sequencing to verify its correctness.
Embodiment 2
[0016] Example 2, protein expression and purification of RuBisCo large subunit gene in vitro
[0017] Transform the recombinants with correct sequencing into Escherichia coli, ER2566, and transfer the overnight bacteria to 100ml LB+Kan50+1% glucose liquid medium at a ratio of 1:100, culture with shaking at 37°C until the OD600 is 0.6-0.8, add 0.1 mol / L IPTG, cultured with shaking at 37°C for 3 hours, sonicated after collection, and detected after SDS-PAGE separation ( figure 2 ), the protein was purified with a Ni column.
Embodiment 3
[0018] Example 3, the background removal effect of western blot hybridization at different developmental stages of rice
[0019] Tissues from the three developmental stages and different parts of rice 93-11 were selected, and the fresh rice material was fully ground with liquid nitrogen to a powder state, which was distributed into pre-cooled centrifuge tubes, and 800 μL of protein lysate (62.5 mmol / L Tris· Cl, pH7.4, 10% glycerol, 2% SDS, 20mmol / L NaF, 2mmol / LEDTA, 1mmol / L PMSF, 5% β-mercaptoethanol), mix quickly and place on ice. Vortex 4-5 times, incubate in ice-water mixture for 10 minutes, centrifuge at 12000r / min at 4°C for 15 minutes, take the supernatant and transfer it to a new 1.5mL centrifuge tube, measure the total protein content of the sample rice by Bradford method, -70°C save. Each sample was loaded with 5 μg of rice total protein, separated by SDS-PAGE, electrotransferred to PVDF membrane, blocked with 5% skimmed milk powder, incubated with antibody for 3 hou...
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Abstract
The invention relates to a reagent for removing nonspecific hybridization of western blot. The invention discloses a method for removing a background of the western blot, belonging to an innovative experiment technology of the field of biological scientific research. The method mainly utilizes a competitive antergic principle to remove or weaken the deep background or the nonspecific hybridization of the western blot due to high-abundance protein in the experimental process of the western blot, and the deep background or the nonspecific hybridization of the western blot can cause the phenomenon that an experimental result has deviation or the expression difference of a target gene in protein levels cannot be really reflected. A recombinant protein is utilized to build a RuBisCo-out kit, the experimental background can be significantly removed or weakened, and therefore the protein expression profiling of the target gene in the different growth stages and different organizational structures of plants can be reflected well.
Description
technical field [0001] The invention relates to experimental techniques in the field of biological scientific research, and belongs to a method for removing background signals in immunoblotting experiments. Background technique [0002] In the Calvin cycle of photosynthesis in C3 plants, Ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCO) catalyzes carbon fixation, thereby making the inorganic Carbon enters the biological chain. The enzyme is a bifunctional enzyme, in CO 2 In the environment with high concentration, ribulose diphosphate carboxylase (RUBP) is carboxylated to act as a carboxylase to form phosphoglycerate; in O 2 In an environment with high concentration, RUBP undergoes an oxidation reaction and acts as an oxygenase to form phosphoglycolic acid and phosphoglyceric acid. As one of the main nitrogen-containing organic compounds in plant leaves, RuBisCo is related to the uptake, utilization and cycle of nitrogen by plants. RuBisCo is also a major storage...
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