Protein Dephosphorylation Protocol
Licia Miller Product Manager
Protein dephosphorylation is an important biochemical process in cells, involving the removal of phosphate groups from specific amino acid residues on proteins. This process corresponds to protein phosphorylation, and the two together form a dynamic balance that is crucial for cell signal transduction. Protein phosphorylation and dephosphorylation, as a pair of reversible biochemical reactions, finely regulate the structure and function of proteins and various physiological activities in cells through the "molecular switch" method.
The main function of protein dephosphorylation is catalyzed by a class of enzymes called protein phosphatases, which can hydrolyze phosphate bonds, i.e. dephosphorylation of serine, threonine and tyrosine sites. This process is crucial for regulating protein activity, stability, subcellular localization and interaction with other molecules.
Disorders in protein dephosphorylation are closely related to the occurrence and development of many diseases, including diabetes, obesity, tumors, neurodegenerative diseases, and autoimmune diseases. Therefore, protein phosphatases have become potential targets for drug discovery. In biomedical research and drug development, understanding and regulating the protein dephosphorylation process is of great significance for revealing disease mechanisms and developing treatment strategies.
To demonstrate the specificity of the antibody for the protein in its phosphorylated state, the protein can be dephosphorylated prior to SDS-PAGE or after transfer to the membrane. Dephosphorylated samples should show little or no staining compared to untreated samples.
Required Materials
- Calf Intestinal Alkaline Phosphatase (CIP)
- CIP buffer: 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol, pH adjusted to 7.9 at 25ºC
- Protease Inhibitor Cocktail, EDTA-Free
Experimental Steps
- Dephosphorylation of lysates prior to SDS-PAGE
1. Lyse cells or homogenize tissue in lysis buffer, then determine protein concentration and save two aliquots of lysate/homogenized tissue. One for dephosphorylation and one for control (untreated sample).
Note: It is generally recommended that the crude extract contains 20-30 µg of protein per lane.
Avoid using sodium orthovanadate (a component of RIPA lysis buffer) and EDTA in the lysis buffer: 10 mM sodium orthovanadate can inhibit CIP (10 units) has 90% activity , while 50 mM EDTA can inactivate CIP activity (10 units) by almost 100%.
If crude extracts are used, protease inhibitors must be included in the CIP buffer.
2. Resuspend the protein/lysate in CIP buffer containing 1 µg protein per 1 µL of 1× buffer with protease inhibitors and without EDTA.
3. Add CIP enzyme to the "+phosphatase" treated samples, adding 1 unit of CIP per µg of protein. Incubate at 37°C for up to 60 minutes. If you are concerned about proteolytic degradation, you can shorten the incubation time appropriately or incubate at a lower temperature.
4. At this point, the sample can be frozen and stored, or proceed with the usual SDS-PAGE procedure.
Note: If desired, sodium phosphate (pH 7.4) can be added as a competitive inhibitor to a final concentration of 10 mM.
- Dephosphorylation after transfer
For antibodies that bind to their phosphorylated protein targets only when the protein is denatured, it may be better to treat the membrane with phosphatase after transfer rather than treating the undenatured lysate with phosphatase before SDS-PAGE. For a good control comparison, the membrane treated with phosphatase should be a piece cut from a single gel blot containing one or more replicate lanes.
Experimental Steps
1. Transfer the gel proteins to nitrocellulose or PVDF and block the membrane with TBS solution containing 0.1% Triton X-100 and 5% BSA at room temperature for 1 hour.
Note that it is not recommended to substitute milk for BSA as milk contains casein, a phosphorylated protein that may produce background staining if the antibody cross-reacts with phosphorylation sites.
2. After the transfer is complete, use a marker or razor blade to draw a line on the membrane to ensure that each sample and its replicates are included in different membrane segments.
3. Then cut the membrane along the scribe line so that each segment contains at least one sample and its corresponding replicate. One fragment can be used for dephosphorylation treatment, while the other fragment can be used for conventional detection to compare the difference before and after treatment.
4. Put the two tablets into separate containers filled with CIP buffer, 3-5 mL in each container.
5. Add CIP to the solution for dephosphorylation.
For more product details, please visit Aladdin Scientific website.