2 Dimensional Gel Electrophoresis (2D GE)

The In Preparation for running a 2D GE, purified proteins must be suspended in a lysis bufer.

For a sharp 2D gel, with proteins of interest in high abundance, use the following buffer:

For additional protein solubility and for proteins of interest in a low abundance use the following buffer:

• Click for the full 2D GE protocol for 7cm or 13cm gel strips.

Ettan spot picker

The FPF strongly recommends excision of protein bands or spots by the robotic spot picker when using a visual stain (e.g. Coomassie or Silver stain).  This will insure optimal spot size and reduce sample loss during automated in-gel digestion.

If you choose to cut the bands manually, the gel pieces must be cubes between 1 to 1.5 mm on all sides including the thickness of the gel, when using 10 or 12% polyacrylamide gels.   If your gel is less than 10%, or less than 1 mm in thickness, you need to cut your gel manually.   Gel pieces need to be equivalent to the 12% gel after dehydration in order to reduce the sample loss.  For example, if you have a 6% gel, the gel spot should be around 3mm cube.  Gel pieces that are too small may become lost in the pipette tips, likewise the gel pieces that are too big may clog the pipette tips.  Sending the correct sample size is your responsibility.

For fluorescent stained gel (e.g. Sypro Ruby stain) gels must be backed onto a glass plate, with the Bind-Silane, picked manually, or restained visually.

If you require more information regarding these procedures, contact the facility staff for more information.

Automated In-Gel Digestion

After the gels are picked or cut and reeady for protein in-gel digestion, you can submitt the samples to the Facility staff (Submission form).

The Facility typically utilizes trypsin, however, if a different enzyme is required, the user must specify it in the submission form and provide the enzyme of choice.

Generally, protein in-gel digestion is a three day procedure.  Included in the process are gel de-staining, rehydration, reduction, alkylation, digestion, peptide extraction, and lyophilization.