跪求bio-rad电泳仪的安装说明

(1) SDS-PAGE gel preparation
SDS-PAGE gel can refer to some literature data preparation, SDS-PAGE gel can also use Pik Wan days production preparation kit (P0012A). The kit provides all the reagents except water and with plastic ware and formula of various concentrations of SDS-PAGE. (mainly divided into concentrated gum and gum, formula can access their own check)
(2) the sample processing
Adding proper amount of [V1] in the collection of the protein concentration in the sample of SDS-PAGE protein sample buffer. For example, 2X or 5X SDS-PAGE protein sample buffer. The use of 5X SDS-PAGE protein sample buffer can reduce the volume of sample, can sample more protein samples in the same volume of sample hole. 5X SDS-PAGE protein sample buffer can be a reference to the relevant documents preparation, can also use the Pik Wan days production of SDS-PAGE protein sample buffer (5X) (P0015).
100 ℃ or boiling water bath heating 3-5 minutes, in order to fully denatured protein [V2].
(3) the sample and electrophoresis
Cooling to room temperature, the protein sample directly onto the SDS-PAGE rubber sample adding hole.
In order to observe effect of electrophoresis and trans membrane effect, and determine the protein molecular weight, it is best to use a prestained protein molecular weight standard (P0066).
SDS-PAGE electrophoresis electrophoresis electrophoretic liquid can use Pik Wan days production (P0014A/P0014B).
Electrophoresis is usually recommended for use in low voltage constant voltage electrophoresis in the upper gum, the use of high voltage constant voltage electrophoresis in bromophenol blue into the lower gum. For the standard Bio-Rad electrophoresis device or similar electrophoretic apparatus, low voltage can be set in the 80-100V, high voltage can be set at about 120V. SDS-PAGE can use electrophoresis ordinary can meet the requirements, can also use the multifunctional electrophoresis Pik Wan days (timing) (EEP102). In order to electrophoresis convenience, can also be used in the whole process of SDS-PAGE constant voltage mode, usually the voltage settings in 100V, and then set the timing for 90-120 minutes. Set the timer can avoid electrophoresis often head.
Usually electrophoresis bromophenol blue to glue at the bottom of the nearby stops electrophoresis, or according to the electrophoresis of prestained protein molecular weight standards, is expected to target proteins have been appropriate separation can be stopped after electrophoresis.
The 3 transmembrane (Transfer) [V3]
We recommend using PVDF membrane in the Western experiment (FFP30/FFP33). The cellulose nitrate film (NC film) (FFN06/FFN09) can also be used, but the cellulose nitrate film more brittle, during operation, especially easy to split the process with tweezers. Steps to use film please refer to manufacturer's recommendation.
Usually if the membrane device using standard wet Bio-Rad, can set the transmembrane current is 300-400mA, transfer film for 30-60 minutes. Can also transfer film in 15-20mA. You can also use the multifunctional electrophoresis Pik Wan days to film (timing) (EEP102). Specific transfer film to the time according to the objective protein size, molecular target protein amount bigger, need more time to film, molecular target protein amount is small, the shorter the time needed to film.
The membrane process, especially the high current fast transfer film, often have a fever phenomenon is very serious, best to turn the membrane tank placed in ice bath to film.
Trans membrane effect can be observed by the use of prestained protein molecular weight standards, usually the largest molecular weight of 1-2 bands are difficult to film. Trans membrane effect can also stain with Li Chunhong (P0022) on membrane staining, to observe the actual transfer film effect. Can also use the Coomassie brilliant blue fast staining solution (P0017) to complete the transfer film of SDS-PAGE gel staining, to observe protein residues.
4 closed (Blocking)
Transfer film is finished, the protein film on the Western washing solution prepared immediately (P0023C) for 1-2 minutes, rinse, to wash away the membrane to membrane liquid. All the steps of the shift from film after film, must pay attention to moisture, avoid the film drying, otherwise easily produce higher background [V4].
Mini desktop vacuum pump (EVAC06/EVAC07) or a drip tube suction washing liquid, adding Western blocking solution (P0023B), moved slowly in the shaking, closed for 60 minutes at room temperature. For the antibody [V5] some of the background is higher, can be 4 ℃ closed for the night. In the whole process of Western we recommend the use of Pik Wan days side swing table (ESHK02) or similar instrument, swing speed side slowly, but also easy to let the solution covering membrane.
The 5 resisting incubation (Primary antibody incubation)
Reference manual one anti Western, anti diluted with an appropriate proportion (P0023A) of an anti dilution.
Mini desktop vacuum pump or dropper, suction sealing liquid, immediately joined the dilution of a good anti, room temperature or 4 ℃ in the side swing slowly shaking table were incubated for an hour. If resisting incubation effect can be an hour, 4 ℃ slow shake overnight incubation. Or according to antibody that select the appropriate incubation temperature and time.
Recovery of an anti [V6]. Join the Western washing liquid (P0023C), in the side of the pendulum table slow shake washing 5-10 minutes. Suction washing liquid, then add the washing liquid washing 5-10 minutes. A total of 3 times of washing. If the higher background may be appropriately extended washing time and washing times.
Note: the Western results usually provide a reference as control, usually can use Tubulin antibody (AT819) or Actin antibody (AA128), for internal testing.
6 two antibody incubation (Secondary antibody inucubation)
Reference two anti instructions, Western two anti diluted with an appropriate proportion (P0023D) diluted horseradish peroxidase (HRP) labeled two anti. Two - according to a resistance to choose, for example, a resistance is small