I. Overview
Western Blot is an immune reaction using antigen and antibody. The protein is first separated by SDS-PAGE gel electrophoresis, and then the protein on the gel is transferred to the solid phase carrier by the action of electric field force, and then the antibody is formed into an antigen-antibody complex. The object is then displayed on the film or film using the principle of luminescence or color development.
The high resolution of SDS-PAGE electrophoresis and the specificity and sensitivity of solid phase immunoassays ensure the accuracy of the final experimental results. WB is a semi-quantitative experiment, which has data analysis compared to qualitative experiments, but requires strict reference standards, so it is not an accurate quantitative relationship, which is also different from quantitative tests.
Second, the technical process
1. A method for selecting an appropriate extracted protein according to the position of the target protein in the cell (in the nucleus, in the cytoplasm, on the cell membrane) and the properties of the target protein (denaturing and non-denaturing)
2. Determination of protein content by suitable methods such as Bradford or BCA
3. Make a suitable concentration of SDS gel according to the size of the target protein.
4. Select the appropriate electrophoresis voltage and electrophoresis time according to the size of the target protein.
5. Select the appropriate film current and film transfer time according to the size of the target protein and the size of the gel to be cut.
6. According to the nature of the target protein, choose a suitable blocking method, such as skim milk powder, BSA and other methods to close
7Select the appropriate concentration of primary antibody for incubation, wash the membrane
8Select the appropriate concentration of secondary antibody for incubation, wash the membrane
9 ECL illuminating liquid for exposure 3. Customer notice
1 test sample: fresh tissue or cells
2 Sample size: The minimum cell content of an indicator for detecting a cell sample is 1*106, and the lowest tissue sample for detecting an index of a tissue sample is 30 mg (recommended to provide more samples for easy grinding)
3 sample storage:
(1) Fresh tissue samples: rapid freezing.
(2) Suspended cells: After collecting, wash the cold PBS 3 times, absorb the residual liquid, and store it frozen.
(3) Adherent cells: After washing the medium, wash the pre-warmed PBS 3 times, collect the trypsin, wash it with cold PBS for 3 times, absorb the residual liquid, and store it frozen; for the unsuitable collection with trypsin The cells (for example, trypsin treatment will digest a part of the protein or have a stimulating effect on the target protein), and after washing the pre-warmed PBS for 3 times, the cells are collected by cell scraping, and the residual liquid is sucked up and stored frozen.
4. Please tell us in advance: the species (human, rat, mouse, rabbit, pig, etc.) of your sample, the number of samples, and the indicators to be tested.
5, sample mail: into the ice bag or dry ice in time to send, it is recommended to choose SF Express land transport or Zhongtong express land transport.
Fourth, the report content and standards
1 target photo exposure photo
2 electronic version of the target protein and internal reference photos
3 gray analysis data
4 protein content data
5 complete experimental report (test method, test parameters, experimental instrument parameters)
5. Fees and service cycle charges and service cycle vary according to the samples provided by the customer. For details, please contact the customer service specialist or refer to the signed cooperation contract.
Sixth, good quality assurance experimental platform system, advanced equipment and high-quality service team engaged in WB experiment for many years to protect your final satisfactory experimental results.
VII. After-sales service The company will conduct customer return visits from time to time to improve the quality of our services. This clause is based on the cooperation contract signed, and the details will vary depending on the contract.
Experimental content: The components separated by electrophoresis were transferred from a gel to a solid support and detected as a probe with a specific reagent for a specific amino acid sequence. The probe used in Western is an antibody that specifically reacts with an epitope presented by a target protein attached to a solid support. The role of this technique is to identify and identify certain specific proteins in a complex mixture of non-radiolabeled protein components.
Eight, prepare the instrument:
Instruments: pressure cooker, glass homogenizer, high-speed centrifuge, spectrophotometer, -20 ° C low temperature refrigerator, vertical plate electrophoresis transfer device, constant temperature water bath shaker, multi-purpose bleaching shaker.
Reagents: single detergent lysate, 0.01mol/L PBS (pH7.3), 10% separation gel, 4% enrichment, G250 Coomassie brilliant blue solution, 0.15mol/L NaCl solution, 2X (5X) SDS loading Buffer, electrophoresis buffer, transfer buffer, 10X Lichun red dye solution, blocking solution, TBST, TBS, elution antibody buffer, developer, fixer, antibody, chemiluminescent reagent.
Miscellaneous goods and consumables: various specifications of tips, centrifuge tubes and samplers; various specifications of beakers, measuring cylinders and other glass equipment; nitrocellulose membrane, latex gloves, plastic wrap, enamel plate (> 20 × 20cm) , X-ray clips, X-rays, one long and one glass rod, timer, absorbent paper.
Western blotting is one of the most widely used techniques in protein detection technology due to its simple operation and high sensitivity. Enter the Shanghai Jinma Project classroom. Teach you no, you don't know.
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