Principle of Ecl color rendering: Luminol can be used as both a marker and a substrate for peroxidase in immunoassays. In the Ecl substrate, containing H2O2 and luminol, under the action of HRP (horseradish peroxidase), it emits fluorescence.
experiment procedure:
1) Mix the two chromogenic substrates 1:1 in equal volume (usually 1ml/membrane each).
2) Cover the mixture on the surface of the film, 1-2 minutes, and shake to make it uniform.
3) Wrap the film with plastic wrap and put it in the splint.
4) Cover the X-ray film on the film in the dark room, clamp the clip, and expose for 1min.
5) Development and fixing.
6) Adjust the exposure time and exposure area according to the result to get the best result.
Note: The fluorescence will become weaker after a period of time.
Remember to operate gloves throughout the whole process, one is to avoid fingerprint contamination affecting the results, and the other is to protect yourself (uncross-linked acrylamide, formaldehyde, etc. are not immediately fatal, but they will slowly poison your body)
1. If there is no reference material before WB – for example, if you don’t know whether there is expression, for example, if you have less antibody, you need to touch the conditions (dilution), you can use the leftover scraps of the shearing membrane to do a few sets of dot hybridization conditions, saving Save some time and save some reagents;
2. After removing the stacking gel, the pre-stained marker can be used to identify the upper and lower directions of the gel and the front and back of the membrane (if the pre-stained marker is used according to the instructions, it is possible that no bands can be seen during electrophoresis, but there is concentration during transfer to the membrane. You can see the effect and the background is white. If you want to see the electrophoresis, you should refer to the amount of electrophoresis, or add 1-2 times more); if the target protein is small, the indicator can also indicate the direction, but if the protein is large, The indicator has already run out, we must pay attention to the up and down direction of the glue, and cutting a small corner is a common method. It is best to cut the membrane and filter paper together (it is not easy to cut the membrane without the filter paper, and the nitrocellulose membrane is easy to crack. It is easier to use a sharp knife + ruler + thick newspaper to cut) as much as possible. Try to be the same size as the glue, and rinse the glue with pure water After equilibration with electrophoresis buffer. When I usually cut it myself, I deliberately make the length and width 1mm less than the glue, so that the film and glue will not touch the filter paper behind each other.
3. For very small proteins, tricine SDS-PAGE electrophoresis can help to improve the resolution of protein size between 1KD-20KD, without glycine, and the concentration of acrylamide does not need to be too high (refer to the 2004 Chinese Journal of Bioengineering for an article. Article (Tricine-SDS-PAGE Method for Efficient Separation of 1kD Small Peptides)
4. The gel should be equilibrated in the transfer buffer before transfer to prevent the gel from deforming, and it will also help to further remove impurities that may hinder the transfer. Some people soak in this step to help the protein renaturation, and the protein activity can be detected directly on the gel.
5. The membrane is floated on the water surface (or methanol level) to allow the liquid to seep up from the bottom through the pores on the membrane to drive away the air in the membrane. After the membrane is thoroughly soaked, the color will become darker, and any white spots or spots are not completely soaked. The logo will affect the transfer of the film. Finally, immerse in buffer to equilibrate. Do not treat PVDF with methanol for more than 15 seconds. Do not allow the membrane to dry out in subsequent steps.
6. Tris glycine system is usually used as the electrotransfer buffer. If some samples need to be sequenced after membrane transfer, CAPS buffer is best used to reduce the contamination of glycine on sequencing.
7. Semi-dry electrotransfer (Semi-Dry) replaces the traditional transfer tank with filter paper saturated with buffer, which saves reagents and has good effect. Since there is no need to “bubble” in the buffer, semi-dry transfer can not only use a homogeneous buffer system, but also a non-uniform transfer buffer system. Semi-dry transfer can be completed in 30 minutes (current 2.5-3.5mA per square centimeter, constant current, cold storage. If the current requirement is small, it can be extended to 60-90 minutes, but to prevent overheating. If there is such a temperature sticker, The reference temperature can be attached), even the transmembrane efficiency of proteins as large as 205KD is as high as 80%. The transfer efficiency was OK for proteins of all sizes. In semi-dry rotation, 2 pieces of glue + film can be stacked up and down together (the area is still calculated according to a single one), as long as the current intensity per unit area is well controlled to prevent overheating.
8. Some people think that adding SDS to the membrane is helpful for the transfer of macromolecular proteins to the membrane. I personally have reservations, because SDS and other detergents affect the binding of nitrocellulose membrane and protein, which is not good.
9. If only Western Blot is performed, the membrane can be stained with Ponceau S and photographed before destaining, which has little effect on the subsequent immune response. Do not stain the membrane with Coomassie brilliant blue or amino black as this will affect the results. If there is a pre-stained marker, you need to use a needle or pen to record the position of the strip, so as not to wash off later and unable to judge the result. Pre-stained markers are also helpful for judging the efficiency and condition of membrane transfer. If all markers larger than the target molecule have been transferred, it is OK.
10. Some people say that electrophoresis under neutral conditions is helpful for protein sequencing. If you want protein sequencing, you need to pour the gel one day in advance, and say that it is pre-electrophoresis for 6 hours (with a reducing agent such as Glycolate) before pouring the layered gel. In addition to HPLC analysis, Ponceau S should be used instead of Coomassie brilliant blue or amino black staining. Others such as sequencing or PTH can choose Coomassie brilliant blue or amino black with higher sensitivity.
11. The PVDF membrane after transfer can be seen faintly without staining in 20% methanol solution under white transmitted light without staining. Sometimes this fast method can identify the bands without staining and avoid the influence of the stained membrane. follow-up experiments.
Note for closure after transfer:
12. After transferring the membrane, other blank spots on the membrane need to be blocked with blocking solution. The sensitivity of Western is somewhat limited by how well the blocking is done. Skim milk is the most commonly used economical formula. Using this blocking agent may cause background contamination due to trace amounts of biotin and alkaline phosphatase, which is not suitable for the detection method of biotin-avidin. Suitable for alkaline phosphatase detection (AP) method. If an alkaline phosphatase detection system is used, the blocking agent should preferably be heated with 6% casein + 1% polyvinylpyrrolidone + 10mmol/L EDTA phosphate buffer for 1 hour at 65 degrees to ensure the inactivation of alkaline phosphatase (add 0.05% stack sodium azide, fresh is best).
Economical skim milk formulas are less compatible with AP, and coupled with a wider substrate choice for HRP, HRP is now an increasingly common choice. However, sodium azide (NaN3) has an inactivation effect on horseradish perazidase (HRP). If the HRP detection system is used, it is better not to add sodium azide to the blocking solution. Remember: the blocking time and the amount of blocking agent must be sufficient.
13. If AP is used as the color development method, Tris buffer system should be selected when blocking, and PBS should not be used, because PBS interferes with AP.
