In-Gel Western Blot Analysis

Required Materials

Load and run the samples on an SDS-page gel following standard electrophoresis procedure.
After electrophoresis, remove the gel gently from the cassettes, and place into 40ml of the fixing solution. Incubate for 10 minutes at room temperature under gentle shaking.
Remove the fixing solution, and wash the gel with DI water 2x 5 minutes. Rinse 3 times with DI water.
Dilute the primary antibody according to supplier's instructions. Incubate the gel in 20ml of primary antibody solution for 1.5 hours at room temperature (or overnight at 4°C).
Remove the primary antibody solution, and wash the gel 2x 5 minutes in 1X TBS. Rinse 3 times with 1X TBS.
Incubate the gel in 20ml of Evident's EviFluor secondary conjugate solution for 1.5 hours at room temperature. Dilute EviFluor secondary conjugate to 1:1000 in 1X TBS (add 20µl of EviFluor to 20ml of 1X TBS). Note: 1:1000 dilution recommended for 600 and 620nm EviFluors.
Remove the EviFluor solution and wash the gel 2x 5 minutes with 1X TBS. Rinse 3 times with DI water.
Image the gel on a suitable imaging system, such as Bio-Rad VersaDoc 4000.

Too much secondary conjugate; reduce the concentration of the conjugate (1:1000 is our recommended dilution).
Insufficient solution for incubations may lead to uneven background. Use enough solution to allow the gel to move freely during the incubation and washing steps.
Washing time is not enough. Extend wash times or increase number of washes. Background may decrease.

The antigen concentration may be too low in the sample. Concentrate the sample and/or increase the sample loading volume.
The concentration of primary antibody is too low. Increase the primary concentration at the labeling step.
The primary antibody dilution buffer is not optimal. Try a different dilution buffer.
The fluorescence intensity of secondary conjugate is not strong enough. Check the fluorescence intensity.
Gel type is not optimal. We recommend Bio-Rad pre-cast gel. Other gel sources may show reduced sensitivity.

Sample overloading. Overloading of the gel is one of the most common reasons for "ghost bands". A dilution series of the starting material usually clarifies which of the bands are from the target proteins.
Primary antibody concentration is too high. Lower the primary antibody concentration.
Primary antibody is not pure enough. Try to use affinity purified antibodies.