Western blotting,which is also called immuno blotting, identifies with specific antibodies proteins that have been separated from one another according to their size by gel electrophoresis.

The procedures from BIOLINK are as follows:

Preparation of lysate

preparation lysate from cell culture

Place the cell culture dish in ice and wash the cells with ice-cold PBS,drain the PBS,then add 0.1mL~1mL lysis buffer with protease inhibitor to it and brings out membrane protein,nuclear protein or total protein we are expected of.

preparation lysate from tissues 

At first ,we have to prepare enough tissue sample,which is fresh or store in an appropriate manner(very critical).Then add 1mL lysis buffer with protease inhibitor to it and after homogenization,it brings out membrane protein,nuclear protein or total protein we are expected of.

Determination of protein concentration

Determine the protein concentration by performing the BCA assay or Braford assay.

SDS-PAGE

Loading the samples precisely in a narrow well by using special gel loading tips or micro-syringe,then run the gel for the recommended time as instructed by the manufacturer,this can vary from machine to machine.

Transfer of protein

In wet transfer, the gel and membrane are sandwiched and all are clamped tightly together after ensuring no air bubbles have formed between the gel and membrane as instructed by the apparatus manufacturer Biorad.And we transfer the protein overnight at 4 deg C and the electric current is 30mA.The conditon needs to be optimized, since it vary from protein to protein. The membrane applied to this experiment can be PVDF or  nitrocellulose membrane.

Blocking the membrane and incubation with the antibody

Blocking the membrane prevents non-specific background binding of the primary and/or secondary antibodies to the membrane by incubating with the 5% BSA solution for 1 hour.

Incubation with the primary antibody for 1.5 hour at room temperature.

Incubation with the HRP-conjugated secondary antibody and HRP-conjugated antibiotin antibody for 1 hour at room temperature,the HRP-conjugated GAPDH/beta-actin may also determine the concentration of GAPDH/beta-actin.

Detection

Detection the protein with ECL kit.

Manual film development is traditionally used and enables the scientist to control the incubation time of the x-ray film in the developing agent and fixation agent.

The camera detects the chemiluminescence emanating from the membrane, transforming the signal into a digital image for rapid analysis with software provided with the detection machine.

Data analysis

Use the Image software to convert the image to a grayscale image and analyze the data.

Experiment Report

We will provide the report with detailed protocols and the images and the data related to the experiment. 

The materials customer should provide

 samples, positive and negative control and the reference

Sample requirements

cell culture, tissue fresh or stored at low temperature(e.g. -20 degC,-80 degC)