Shanghai Jinma Biological Summary: WB is a shorthand for Western blotting. The ultimate result of a gene expression is the production of the corresponding protein (or enzyme). Therefore, detection of proteins is a major indicator of gene expression. There are many methods for detecting proteins. In addition to ELISA, blotting methods similar to those for detecting DNA and RNA can be used. The first two methods have the meaning of "South" and "North", so this method is extended to be called Western (Western) blotting method, which can distinguish the specific antiserum by SDS polyacrylamide gel electrophoresis. A sex protein. The protein resolved on the polyacrylamide gel was transferred to a nitrocellulose membrane and co-incubated with the primary antibody. The first antibody specifically binds to the antigenic determinant of the egg to be isolated, and then the antibody that has been bound is detected by another egg, such as 135I-protein A or horseradish peroxidase-conjugated goat anti-IgG. This method takes 6 hours or overnight. And our customers often make the following problems when doing WB experiments? What are they? Let's follow the Xiaobian together to see the latest analysis of the 15 common problems of Western blotting by Shanghai Jinma Bio:
1. Do the western blot at the cell level, how many cell extracts are enough for western blot?
Answer: Generally 5×10^6 is enough
2. If the target protein is a membrane protein or a cytosolic protein, what should you watch out for?
Answer: If it is a membrane protein and a cytoplasmic protein, the detergent used should be much milder.
NaF deactivates the activity of phosphorylase.
3. The protein to be separated is 260kd in molecular weight. What is the appropriate concentration of separation gel for SDS-PAGE electrophoresis? What is the concentration of the laminated glue? Is such a large molecular weight protein easy to use as a Western Blot?
Answer: 260kd protein is not good, 6% separation gel, Stacking Gel 3.5%. Can also use Kingsley's pre-made glue
4. How long can the protein be stored after denaturation?
Answer: -80 ° C, no problem for one or two years. The two most important two: don't be hydrolyzed by proteases; don't digest the bacteria (it is also hydrolyzed by the enzyme).
5. When doing the western sample of the tissue sample, what is the flaw in the sample processing? Also, have you used large bovine serum as a sealer? What is the concentration? Is the effect better than BSA?
Answer: Grinding, homogenization, and sonication are required. The solubility of the protein is better, the centrifugation is sufficient, the membrane protein needs to be extracted by a more violent method, and the low-abundance membrane protein may be extracted step by step (ultracentrifugation). Another point is that the protease activity in the tissue is stronger, and it is necessary to pay attention to inhibiting the activity of the protease (adding PMSF and protease inhibitor cocktail), and the blocking agent is generally used as 5% skim milk powder. If the primary antibody is a polyclonal antibody, BSA is also a good choice.
6. Can immunohistochemistry and Western Blot use the same antibody?
Answer: When an antibody is formed, the antibody recognizes an antigenic determinant (also known as an epitope) that has not been denatured. Some epitopes are linear, while others are conformational; linear epitopes are not affected by protein denaturation, natural Both proteins and boiled proteins are contained; conformational epitopes are lost due to the spatial structure of the protein and post-cooking denaturation. If the antibody you are using recognizes several amino acids in a protein, that is, a linear epitope, then the antibody can be used for both immunohistochemistry and Westernization, and if the antibody recognizes a conformational epitope, it can only be used. Immunohistochemistry. The amino acid range indicating the recognition of such antibodies is indicated in the general antibody specification. (limited to monoclonal antibody)
7. Repetitive application of antibodies in Western Blot: Antibody working solutions generally do not claim to be stored repeatedly, but if the antibodies are more precious, they can be used 2-3 times. It should be used within 2-3 days after dilution and stored at 4 degrees to avoid repeated freezing and thawing.
8. How to identify NC film\PVDF film\Nylon film?
Answer: Nylon membrane is an ideal nucleic acid solid phase support. There are many types; nitrocellulose membrane is the most widely used solid phase support at present, and the price is the cheapest; PVDF membrane is somewhere in between.
In terms of binding ability: nylon membrane can bind DNA and RNA up to 480-600μg/cm2, and can bind nucleic acid fragments as short as 10bp; nitrocellulose membrane can bind DNA and RNA up to 80-100μg/cm2, for 200bp The binding ability of nucleic acid fragments is not strong; the ability of PVDF membrane to bind DNA and RNA can reach 125-300μg/cm2.
In terms of temperature adaptability: after the nylon membrane is baked or irradiated with ultraviolet light, a part of the pyrimidine base in the nucleic acid can bind to the positive charge on the membrane; the nitrocellulose membrane binds to the DNA by hydrophobic interaction, and the binding is not strong; PVDF The membrane is firmly bonded and resistant to high temperatures, and is particularly suitable for Western blotting.
In terms of toughness: the nylon membrane is strong; the nitrocellulose membrane is brittle and easily broken; the PVDF membrane is stronger.
In terms of repeatability: the nylon membrane can be repeatedly used for molecular hybridization. After hybridization, the probe molecules can be eluted by alkali denaturation; the nitrocellulose membrane cannot be reused; the PVDF membrane can be reused.
9. What is the purpose of soaking PVDF membranes with methanol when doing Western Blot?
Answer: The purpose of the PVDF membrane with methanol bubbles is to activate the positively charged groups on the PVDF membrane, making it easier to bind to negatively charged proteins. This is also the purpose of adding methanol to small molecules.
10. What is the reason why the glue used in running electrophoresis is always "shrinking"? Is there something wrong with it?
Answer: No problem, the moisture in your glue is evaporated. Wrap the plastic wrap over the night and add some water to keep the humidity inside. If it is overnight, the moisture in the gel is evaporated, and it can be wrapped in plastic wrap. It may also be a problem with the mother liquor (30% polyacrylamide). You can re-form an observation; replace the reagents, try to change them, choose good. Reagents to avoid trouble finding problems. Too high a level of methanol in the decolorizing solution can also cause gelation.
11. Can't transfer large molecular weight proteins to the membrane very well, how to solve the problem of low transfer efficiency?
Answer: Consider: adding 20% ​​methanol to the transfer buffer (refers to the final concentration) (optimized transfer buffer, refer to the Protein Technology Manual), because methanol can reduce protein elution efficiency, but can increase protein and NC Membrane binding ability, methanol can prevent gel deformation, methanol can prolong the transfer time for high molecular weight protein; transfer buffer is added to the final concentration of 0.1% SDS, also to increase transfer efficiency; use high quality transfer film, or use small pore size NC Membrane (0.2 micron); cross-linked with glutaraldehyde; low-concentration gel, as low as 6%. If too large, you can also consider using agarose gel; increase the transfer voltage / current; increase the transfer time.
12. What kind of dyeing is Western Blot?
Answer: (1) Anionic dyes are the most commonly used, especially amino black, which is fast decolorizing. The background detection limit can reach 1.5μg. Although the horse has the same sensitivity as amino black, it has slow decolorization and high background. Ponceau S and Fast Green are easily removed from the protein after detection for subsequent amino acid analysis. The disadvantage is that methanol in the solvent system can cause shrinkage or destruction of the nitrocellulose membrane. Cannot be used for positively charged films. Low sensitivity.
(2) Colloidal gold, high sensitivity, detection range can reach pg level, but the dyeing ratio is stable.
(3) The biotinylation sensitivity is between 1, 2 and can be used for any kind of membrane.
13. Is the current used in the film transfer accurate than the voltage, is it based on 0.8mA/cm2, usually about 1 hour?
Answer: No, the semi-dry method recommends constant current, and the current and time are generally determined according to the size of the protein of interest.
14. What is the purpose of adding methanol?
Answer: Adding methanol has a certain fixed effect, because small molecules of protein are easy to transfer out (especially on nitrocellulose membranes, because NC membranes have weaker ability to bind proteins).
15. What is the principle of PVDF membrane and nitrate membrane binding protein?
Answer: In general, the nitrocellulose membrane is linked to proteins by hydrophobic action. In this case, after repeated washings, the protein is easily dropped and the result is poor. The nylon membrane is mainly bonded by positive charge and protein on its membrane (note: commonly used PVDF, ie, a positively charged nylon membrane), but also has a hydrophobic effect, but is relatively weak. In this case, the PVDF membrane and the protein are more firmly bonded and are less likely to fall off, and the result is better.
Shanghai Jinma WB experimental service process
1. The customer provides primary antibody, preferably imported antibody, the company can purchase
2. Extract the total protein of the sample for total protein quantification
3. According to the results of total protein quantification, fine-tune the difference in concentration between samples, high temperature denaturation, adding loading buffer, loading and running, and color transfer.
4. The obtained film is scanned by software. When quantitative analysis is carried out by relative quantitative method, the reference protein such as housekeeping protein is subjected to the same operation, and the obtained value is used to correct the protein amount, and finally the relative expression of the target protein is obtained. the amount
5. Provide experimental reports: including detailed experimental methods and related charts of western blot results
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