one. Preparation of protein samples
Add the appropriate volume of lysate according to the tissue net weight (g): lysate volume (ml) = 1:10 (add the final concentration of 1 mM PMSF, appropriate concentration of protease inhibitor and phosphorylase inhibitor to avoid The source enzyme decomposes the protein, affecting the extraction of the protein) and homogenizing.
Sample preparation of proteins is the first step in western blotting and a key step, requiring all proteins to be obtained. The following questions should be noted:
(1) At the appropriate salt concentration, the maximum solubility and reproducibility of the protein should be maintained.
(2) Selecting a suitable surfactant and reducing agent to destroy all non-covalently bound protein complexes and covalent disulfide bonds to form a solution of the respective polypeptide.
(3) Try to remove the interference of nucleic acid, polysaccharide, lipid and other molecules. After centrifugation, carefully take the middle layer clear solution, taking care not to absorb the bottom layer precipitate and the upper layer lipid.
(4) To prevent artificial modification of the protein during the treatment, the preparation process must be carried out at a low temperature to avoid cell breakage and release various enzyme modifications. (You can add enzyme inhibitors such as PMSF)
(5) After the sample is prepared, it should be stored separately, but be careful not to freeze and freeze repeatedly.
two. Preparation of gel
For the preparation of uniform gel, a protein with a smaller molecular weight is selected from a larger concentration of gel.
After 30% of the PAG is configured, it is filtered and stored at 4 °C. 10% SDS is stored at room temperature. The AP is unstable and is available when used.
The key to making the gel is the polymerization time. The separation gel polymerization is controlled from the addition of 10% AP and TEMED to the start of the gel for 10-20 min (not completely agglomerated). The optimum polymerization time of the concentrated gel is 8-10 min. It can be controlled by controlling the amount of AP and TEMED. AP, the amount of TEMED is too high, the local gelation is too fast, the gel will be uneven, and the electrophoretic protein bands will bend. When the ambient temperature is high, the amount of AP, TEMED should be reduced. Oxygen in the air also affects the polymerization of the glue, so a layer of water or n-butanol should be added to the separation gel to isolate the oxygen.
three. Electrophoresis common problems:
(1) The phenomenon of “smile” appears in the strip (the middle and the concave sides are lifted up): mainly due to the uneven solidification in the middle part of the gel, which should be electrophoresed after the gel is fully solidified, or fully mixed after the catalyst is added.
(2) The phenomenon of “frowning” appears in the strip (downward on both sides of the middle drum): due to the bubble at the bottom between the two glass plates, the bottom bubble should be excluded.
(3) There is a tailing phenomenon in the belt: mainly because the sample dissolves poorly; the separation gel concentration is too large, and the electrophoresis buffer is placed for too long. Recommendation: Centrifuge before loading, select the appropriate sample buffer, add the appropriate amount of sample to promote solvent; use the newly configured running buffer to reduce the gel concentration or use a gradient gel.
(4) Texture phenomenon in protein bands: caused by insoluble particles in the sample. Recommendation: Centrifuge before loading and add an appropriate amount of solvent.
(5) The indicated bromophenol blue has run out of the bottom plate, but the protein has not run down: it is related to the concentration of buffer and separation gel. Should replace the correct pH buffer, reduce the gel concentration or use a gradient gel
Wet electrotransfer notes:
(1) The pH of the buffer is important in order for the protein component to be efficiently transferred from the gel to the solid phase paper. Some protein bands that are not very large in molecular weight cannot be transferred from the gel even if the electrotransfer time is extended.
On the membrane. This is because the protein is just in the state of isoelectric point in the transfer buffer, so it should be appropriate.
When changing the pH of the transfer buffer to facilitate membrane transfer. In addition, for proteins with smaller molecular weight,
An appropriate amount of methanol is added to the buffer because methanol promotes their immobilization on the solid phase membrane. But for big
Molecular weight proteins, especially basic proteins, are not suitable for methanol in buffers because of methanol
Make the gel pore size smaller, which is not conducive to the transfer of proteins with larger molecular weight, and methanol can bind to basic protein
When the SDS is dissociated and positively charged or electrically neutral, the protein is more difficult to transfer from the gel.
(2) Selection of electrotransfer film: NC film, diazotized film and cationic nylon film, PVDF film. Where NC
The membrane is most commonly used, its protein capacity is large, and it can be detected by various dyeing methods, but it
The binding to the protein is non-covalent and the membrane is very brittle after drying. In addition, the pore size of the membrane is also considered.
An important factor, the protein of 30KD NC membrane with a pore size 0.22um, with more than 0.45um 30KD
The NC membrane of the aperture.
(3) Transfer selection constant current, each transfer tank 150mA, 10KD transfer 40min, 10-30kd transfer 50min, 30-100KD transfer 70min, 100kd transfer 80min, transfer finished Li Chunhong S The film is dyed and the transfer is successful. After the transfer is finished, the NC film is removed and placed in the Lichun Red S solution. The red band can be seen at room temperature for 5-10 minutes, and the red band can be washed by washing with TBS several times. Follow-up experiments.
(1) After electrophoresis, other blank sites on the membrane that do not bind to the protein need to be blocked with a blocking solution to avoid non-specific binding of the primary or secondary antibody. This is because the sensitivity of WB depends largely on the quality of the closure. The long-term closure time (excessive closure) leads to the masking of the antigenic epitope, which reduces the detection sensitivity; the short closure time (not completely closed) leads to the final The background is increased or the signal to noise ratio is too low, so the selection of the blocking solution and the optimization of the conditions are important.
(2) Appropriate amount of preservative should be added to the blocking solution to avoid blocking effect caused by blocking protein denaturation.
six. Antibody hybridization
(1) If there are too many non-specific bands, the concentration of the antibody can be appropriately reduced, the number of washings can be increased, the denaturation time can be prolonged before protein electrophoresis, the amount of protein loaded can be reduced, and the blocking time can be prolonged.
(2) If the signal is weak, the film may be incomplete, the transfer time and current may be optimized, the concentration of the antibody may be increased, and the exposure time may be appropriately extended.
(3) If the background is high, the antibody concentration can be appropriately lowered, the secondary antibody can be filtered, the sealing time can be extended, the rinsing time can be increased, and the exposure time can be reduced.
(1) Film exposure for a few seconds to a few minutes, depending on the intensity of the chemiluminescence strip on the film. The exposure time of the film should not be too long. If the time is too long, the film background will become darker. Flush the film to determine the antigen to be measured
Correct exposure time
(2) The step of rinsing the film: the exposed film is first rinsed in the developer until the band appears, generally
Within 1min, too long will also become darker as the film background. Rinse in the fixer solution for ten seconds.
After the developer has been rinsed, rinse it with water to prevent the components in the fixer from remaining on the film.