(1) Protein separation Separation of protein samples by SDS-PAGE, urea-PAGE, isoelectric focusing (1EF)-PAGE, two-dimensional gel or agarose gel as a medium with a gradient of 5% to 20% The basic operation is the same as gel electrophoresis. The difference is that the thickness of the gel needs to be 0.8mm. The separated gel (except SDS-PAGE) should be immersed in the buffer containing SDS to make the protein negatively charged, so as to facilitate the protein in the gel. The electric field was transferred to the NC film on the positive electrode side. Therefore, when separating proteins, most of them are carried out using an SDS-PAGE system. However, it is also replaced by a non-SDS-PAGE system. When using this system for transfer operation, the protein on the gel needs to be transferred to the two NC membranes at the positive and negative ends (ie, two NCs are placed on the gel). The sensitivity of the procedure is low between the membranes.
(II) Treatment of NC membrane Take an NC paper of the same size as the gel strip (cut with a scalpel) and soak in the electrophoresis transfer buffer (usually 25mmol/L Tris, 191mmol/L glycine, 20% methanol) 0.5h (made in domestic NC paper for more than 2h).
(3) Treatment of the gel sample After electrophoresis in SDS-PAGE, the gel strip segment to be transferred is cut or the whole gel plate is immersed in the electrophoresis transfer buffer for 0.5 h, and gently shaken to remove the gel. SDS, mercaptoethanol and other substances make the ionic strength in the gel the same as the transfer liquid, in order to facilitate the recovery of the natural structure and biological activity of the protein and prevent the gel from expanding.
(4) Prepare sponge and filter paper and cut two pieces of sponge and filter paper of the same size as the NC film, and soak them with electrode buffer.
(5) Production of “Sandwich Cake†Place the plexiglass plate with holes on a clean glass plate, then cover with a sponge, spread a layer of filter paper on the sponge, and then carefully place the gel on the filter paper. The end of the NC film should be placed on the adhesive surface (do not pick it up once it is lowered). A filter paper, a sponge, and a perforated plexiglass plate were sequentially coated on the NC film. After zui, it is tied with a rubber band and placed in an electric field containing an electrode buffer. The end with the gel is placed toward the negative electrode, and the end with the NC film is placed toward the positive electrode. After the power is turned on, the current is adjusted to about 60 mA (constant current), and electrophoresis is carried out for 6-18 hours in a 4 ° C environment. When making a "sandwich cake", there should be no bubbles between every two layers, otherwise it will affect the transfer of the sample. In addition, the electrode solution should contain 20% methanol to prevent gel deformation during electrophoresis. The molecular mass has a long transfer time, and vice versa. The transfer process is also a process that further removes SDS and restores the structure and biological activity of the transferred material.
(6) Identification
1. Enzyme-linked immunosorbent assay (ELISA) (1) Reagents: 1 PBS solution, 0.025 mol/L phosphate buffer containing 0.15 mol/L NaCl, pH 7.4; 2 PBS-T solution, containing 0.3% Tween PBS solution (quenching agent); 3HBP-IgG solution, HRP-human antibody complex solution; 4 biphenyl anisidine solution, 50 mg of biphenyl anisidine dissolved in 10 ml of methanol; 5 staining solution (prepared before use), take Mix 27 ml of bPBS-T solution, 3 ml of biphenyl anisidine solution, and 35 ul of 3% hydrogen peroxide solution.
(2) Staining: The blotted NC membrane was rinsed in 50-200 ml of PBS for 5 min to remove the gel adhering to the NC membrane, and then in PBS-T or PBS-BSA (PBS containing 3% bovine serum albumin) In solution, shake at 37 °C for 15 min (blocking reaction), react with enzyme-labeled solution 20 ml (1; 25, dilution depending on HRP activity) for 2 h (at 37 ° C), then use PBS Rinse with PBS-T or PBS-BSA, respectively. Then, after immersing in 30ml (if the membrane is 4cm×8cm), the staining solution is soaked for about 5 minutes, and the specific antigen brown band can be displayed (note: once the band shows it is taken out immediately), rinsed with distilled water, sealed and stored in distilled water at 4 ° C. in.
2. The NC paper blocked by autoradiography is reacted with a radionuclide-labeled antibody or other corresponding ligand, followed by rinsing, drying, tableting, and rinsing to exhibit an autoradiographic band.
In addition to the above identification methods, staining can also be performed using reagents such as Indian ink, Tianjin drawing ink, amino black, and Coomassie brilliant blue to identify the protein bands imprinted on the NC membrane.
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