Western, also known as Western blot, Western blotting, Western blotting, is one of the important methods for detecting proteins with antibodies. Follow the steps below. 2. Electrophoresis (Electrophoresis) (2) Sample processing (2) It is generally recommended to use low-voltage constant-pressure electrophoresis in the upper layer of gel when electrophoresis, and high-voltage constant-pressure electrophoresis in the case of bromophenol blue entering the underlayer. For the standard electrophoresis device or similar electrophoresis device of Bio-Rad, the low voltage can be set at 80-100V, and the high voltage can be set at about 120V. (3) For the convenience of electrophoresis, the whole SDS-PAGE process can also be used for constant voltage. Usually, the voltage is set at 100V, and then the timing time is set to 90-120 minutes. Setting the timing avoids frequent electrophoresis. Usually, when electrophoresis, bromophenol blue reaches the vicinity of the bottom end of the gel to stop electrophoresis, or according to the electrophoresis condition of the pre-stained protein molecular weight standard, it is expected that the target protein has been properly separated and the electrophoresis can be stopped. (2) The washing solution was exhausted with a micro table vacuum pump, Western blocking solution was added, and the mixture was shaken slowly on a shaker and sealed at room temperature for 60 minutes. For some antibodies with higher background, they can be blocked overnight at 4 °C. 5. Primary antibody incubation Vegetarians Size 2 Empty Capsule Vegetarians Size 2 Empty Capsule,Vegetable Empty Capsules,Vegetable Gelatin Empty Capsules,Hpmc Vegetable Empty Capsule Ningbo Jiangnan Capsule Co., Ltd. , https://www.jncapsule.com
1. Collect protein sample (Protein sample preparation)
The adherent cells, suspension cells or tissue samples are lysed using cell lysates. The protein concentration of each protein sample was then determined.
(1) SDS-PAGE gel preparation
SDS-PAGE gel (separation gel and gel) can be prepared by reference to some literatures.
An appropriate amount of concentrated SDS-PAGE protein loading buffer was added to the collected protein samples. For example, 2X or 5X SDS-PAGE protein loading buffer. The 5X SDS-PAGE protein loading buffer can be used to reduce the loading volume, and more protein samples can be loaded in the same volume of the well. 5X SDS-PAGE protein loading buffer can be prepared by referring to the relevant literature, and heated at 100 ° C or boiling water bath for 3-5 minutes to fully denature the protein.
(3) Loading and electrophoresis (1) After cooling to room temperature, the protein sample can be directly loaded into the SDS-PAGE gel sample well. In order to facilitate the observation of the electrophoresis effect and the effect of the transfer film, and to judge the molecular weight of the protein, it is preferred to use the pre-stained protein molecular weight standard.
3. Transfer (Transfer)
(1) We recommend the use of PVDF membranes in Western experiments. A nitrocellulose membrane (NC membrane) can also be used, but the nitrocellulose membrane is relatively brittle, and is easily cleaved during handling, particularly during the process of gripping with tweezers. Please refer to the manufacturer's recommended procedure for the use of the membrane. Generally, if Bio-Rad's standard wet film transfer device is used, the transfer current can be set to 300-400 mA, and the film transfer time is 30-60 minutes. It can also be transferred overnight at 15-20 mA. The specific membrane transfer time depends on the size of the protein of interest. The larger the molecular weight of the target protein, the longer the membrane transfer time required, and the smaller the molecular weight of the target protein, the shorter the membrane transfer time required.
(2) During the film transfer process, especially when the high current is rapidly transferred, there is usually a very serious heat generation phenomenon. It is better to place the transfer film tank in an ice bath for film transfer. The effect of the transfer film can be observed by using the pre-stained protein molecular weight standard. Usually, the 1-2 bands with the largest molecular weight are more difficult to transfer to the film. The effect of the transfer film can also be dyed with the Ponceau red staining solution to observe the actual film transfer effect. The SDS-PAGE gel that has been transferred can also be stained with Coomassie Brilliant Blue Rapid Stain to observe protein residues.
4. Blocking
(1) Immediately after the film is transferred, the protein film is placed in a pre-prepared Western washing solution, and rinsed for 1-2 minutes to wash away the transfer liquid on the film. From all steps after the film is transferred, it is necessary to pay attention to the moisturizing of the film to avoid drying of the film, otherwise it is easy to produce a high background.
(1) According to the instructions of the primary antibody, the primary antibody is diluted with a Western primary antibody dilution in an appropriate ratio.
(2) Drain the blocking solution with a micro-desktop vacuum pump, immediately add the diluted primary antibody, and incubate for one hour at room temperature or 4 °C on a side-waist shaker with gentle shaking. If the primary antibody is not incubated for one hour, it can be incubated overnight at 4 °C with gentle shaking.
(3) Recovering the primary antibody. Add Western Wash and gently shake for 5-10 minutes on a side swing shaker. After draining the washing solution, the washing solution is added and washed for 5-10 minutes. Washed a total of 3 times. If the result background is higher, the washing time can be appropriately extended and the number of washings can be increased.
6. Secondary antibody inucubation
(1) Diluted the horseradish peroxidase (HRP)-labeled secondary antibody with a Western secondary antibody dilution in an appropriate ratio with reference to the instructions for the secondary antibody.
(2) Drain the washing solution with a micro-desktop vacuum pump, immediately add the diluted secondary antibody, and incubate for one hour at room temperature or 4 °C on a side-waist shaker with gentle shaking.
(3) Recovering the secondary antibody. Add Western Wash and gently shake for 5-10 minutes on a side swing shaker. After draining the washing solution, the washing solution is added and washed for 5-10 minutes. Washed a total of 3 times. If the result background is higher, the washing time can be appropriately extended and the number of washings can be increased.
7. Detection of proteins
(1) Refer to the relevant instructions, use Ingen's Enlight and other high-sensitivity ECL luminescent liquid to detect protein.
(2) X-ray automatic film processor can be used for washing. If there is no automatic processor, you can use the development and fixing kit to prepare the developer and fixer for manual washing.
8. Membrane recovery
If the protein sample is invaluable, the protein can be treated with a Western primary antibody secondary removal solution to re-use the protein membrane.