Protein Dephosphorylation Methods

Dephosphorylation of Proteins Fixed on a Membrane

Materials Required:

• Protein Membrane Blot:
  -PVDF membrane is recommended.
  -Refer to Western Blot Protocol (up to Protein Transfer) to prepare protein membrane blot.
• Lambda Phosphatase Enzyme: Sigma Aldrich product, includes 10X phosphatase buffer and 10X MnCl₂, Catalog number: P9614
• 1X Incubation Buffer: Lambda phosphatase buffer and MnCl₂ solution diluted to 1X with dH₂O
• Alkaline Phosphatase Enzyme: Sigma Aldrich product, Catalog number: P0114
• Conical tube or microtube with screwcap
• Deionized H₂O (dH₂O)
• Wash Buffer (1X TTBS): 14 mM NaCl, 2 mM TRIS, 1%(w/v) Tween 20, pH 7.6
• 100% Methanol

Dephosphorylation of Proteins in a Lysate

Materials Required:

• 10X Lysis Buffer: 100 mM TRIS, 100 mM NaCl, pH 8.0
• Detergent for Lysis Buffer: 10% (v/v) NP40-LB
• 10% SDS: Phosphatase deactivating agent
• Lambda Phosphatase Enzyme: Sigma Aldrich product that includes 10X Phosphatase Buffer and 10X MnCl₂, Catalog number: P9614
• Alkaline Phosphatase Enzyme: Sigma Aldrich product, Catalog number: P0114
• 1X Lysis/Incubation Buffer: Prepared from 10X Lysis buffer, 10X Phosphatase Buffer, and 10X MnCl₂ solution
• Spatula, plastic transfer pipette, and scissors
• Conical tube or microtube with screwcap
• Ice
• Deionized H₂O (dH₂O)
• Heat block: Set to 95°C
• Sonicator: Select probe and optimize strength based on lysis buffer volume and tube size to avoid foaming of the protein sample.

This protein dephosphorylation method uses a two-step approach to focus on specificity and potency. The first treatment is done with a lambda phosphatase to specifically target phosphate groups from threonine, serine, and tyrosine amino acids for dephosphorylation. Most phosphatases only dephosphorylate serine and threonine amino acids. If you are testing the phosphospecificity of a tyrosine phosphospecific antibody, lambda phosphatase is essential. The second dephosphorylation treatment is done with an alkaline phosphatase, a significantly more potent enzyme than lambda phosphatase. The alkaline phosphatase only dephosphorylates phosphate groups from threonine and serine amino acids, and therefore is not intended to be utilized alone to determine the phosphospecificity of tyrosine phosphospecific antibodies. When using both lambda and alkaline phosphatases in conjunction with each other, it is best to separate the treatments so the alkaline phosphatase doesn’t inhibit the activity of the lambda phosphatase. There are a variety of phosphatases one may use to dephosphorylate proteins; the ones listed in this protocol are recommended examples. The end user is highly encouraged to optimize their chosen phosphatase(s) before determining phosphospecificity of an antibody.
Calculations for the recommended examples can be found at the end of the protocol. It is important to know the specific activity of the phosphatase(s) chosen to determine the amount of enzyme needed. If dephosphorylating pure protein and the molecular weight is known, use this number to determine the amount of each phosphatase needed. If you are preparing a lysate with mixed proteins an assumption of the average molecular weight, aka highly educated guess, is best used. The calculated phosphatase volumes are a starting point to determine the amount needed to dephosphorylate the protein. Since the calculation is based on assumption, additional experimental optimization may be needed.

Lysate Preparation

1. Determine the protein mass of the organs or tissue culture cells.
   Tech Tips:
   • Organs: Brush off any debris from frozen organs. Wash fresh organs with 1X PBS and dry with Kimwipe. Weigh fresh or thawed organs. Estimate protein mass as 10% of total mass.
   • Suspension Cells: Make estimate based on cell count.
   • Adherent Cells: This protocol is not recommended for adherent cells due to non-optimal lysing conditions. The ‘Dephosphorylation of Fixed Protein on membrane’ protocol is recommended.
2. Based on the estimated protein mass for the samples, calculate a desired final concentration of lysate.
   Tech Tip:
   • A final concentration below 10 mg/mL is recommended for optimal lysing. Calculate concentration with formula below:
3. Determine the volume needed for the desired protein concentration and prepare 1X lysis/incubation buffer. Chill on ice for 30 minutes before lysing.
   Tech Tips:
   • Bring all lysis/incubation buffer components to a final concentration of 1% or 1X.
   • 10% NP40-LB or 10% Triton are recommended detergents to use in the preparation of 1X lysis buffer. These detergents preserve proteins and lyse most enzymes, but do not lyse phosphatase enzymes.
   • 1% SDS lyses everything, including phosphatase enzymes. It is important not to use 10% SDS prior to dephosphorylation treatment.
4. In an appropriate volume conical tube or microtube, add the calculated amount of freshly prepared chilled lysis buffer to organs/cells to reach the desired concentration. Keep sample chilled on ice. Make a note of the volume of lysis buffer added.
   Tech Tips:
   • Organs: For large organs, cut into 1/4” sections. For organs that are thick and dense, cut even smaller sections.
   • Suspension Cells: Pipette media and cells into conical tube. Pellet cells by centrifugation
     (1 minute at 1800 x g) and remove media. Resuspend cells with 1-3 mLs of room temperature 1X PBS to wash. Repeat centrifugation to pellet and remove 1X PBS. Add desired volume of lysis buffer.
5. Sonicate sample in 5-20 second intervals until solution is clear and can be easily pipetted without clogging. Keep sample on ice while sonicating and after sonication is completed.
   Tech Tips:
   • Organ and cell membranes will appear as white, stringy debris in lysate. It is essential to completely lyse this debris when studying transmembrane proteins.
   • To avoid puncturing/melting the plastic tube, minimize prolonged contact with the probe to the conical tube walls.
   • To avoid foaming, keep probe tip submerged in lysate until sonication is complete.
   • Pipette sample after sonicating. If the lysate clogs while pipetting, sonicate again until clog is no longer present.
6. Divide lysate into 2 equal volumes: Label one the control lysate, which will not be treated with phosphatase enzymes. Label the other the treated lysate, which will be treated with the phosphatase enzymes. Make a note of the volume for each sample.
7. Spike the control lysate with 10% SDS to a final concentration of 1% SDS. Make note of volume of 10% SDS added to the control lysate. Heat control lysate at 95˚C for 10 minutes. Set sample aside, leave at room temperature.

Protein Dephosphorylation

1. Add 1 μl of lambda phosphatase per 1 mg of protein to the treated lysate. Make a note of the volume of enzyme added to the treated lysate. Incubate in a water bath at room temperature for 30 minutes.
   Tech Tip:
   • Testing the dephosphorylated lysate is advised before using the lysate to determine phosphospecificity of an unknown phospho-antibody. Try using a known phosphospecific antibody that is phosphorylated at the same type of residue, i.e. Ser, Thr, or Tyr as your unknown. Since the original calculation was based on assumption further optimization of the amount of enzyme and incubation time may be required.
2. Add 1 μl of alkaline phosphatase per 10 mg of protein to the treated lysate. Make a note of the volume of enzyme added to the treated lysate. Incubate in a water bath at room temperature for 10 minutes.
3. Add 10% SDS to the treated lysate to a final concentration of 1% SDS and place in a heat block at 95˚C for 10 minutes to inactivate the phosphatase enzymes. Make note of volume of 10% SDS added to the treated lysate.
4. Add together the recorded volumes of reagents added to each lysate in previous steps. Adjust the lysates to equal volumes using the freshly prepared lysis buffer that contains 1% SDS.
5. Determine the protein concentration using the control lysate.
   Tech Tips:
   • The prepared 10X buffer for the enzyme used in this protocol contains DTT, this reagent will interfere with the BCA assay. It is recommended to use a different assay to determine protein concentration.
   • It is not recommended to use the treated lysate to determine the protein concentration as the concentration will be higher than the control due to the presence of the phosphatase enzyme.
6. Lysate is ready for assay application or can be frozen at -80˚C for long term storage.
   Tech Tip:
   • When producing a large preparation of treated cells or organs, aliquoting the lysates into small volumes for individual assay applications is recommended for convenience.

Phosphatase Calculation

Important factors, physical constants, and assumptions:

• Avogadro’s Number: 6.023 x 10²³ molecules/mol
• Assumed average molecular weight of the protein lysate: 100,000 daltons (g/mol)
• The Specific Activity of the Phosphatases
  Lambda Phosphatase: 1 Unit hydrolyzes 1 nmol of phosphate per minute; 1 μl of lambda phosphatase contains 400 units, so for every 1 μl of phosphatase 400 nmol of phosphate will be hydrolyzed per minute.
  Alkaline Phosphatase: 1 Unit hydrolyzes 1 mmol of phosphate per minute; 1 μl of alkaline phosphatase contains 133 units, so for every 1 μl of phosphatase 133 mmol of phosphate will be hydrolyzed per minute.
• If you know the exact size and amount of that pure protein, use those numbers. If not, don’t freak out! An assumption (aka highly educated guess), is best used in these scenarios. The phosphatase volumes calculated are starting points to determine the amount needed to dephosphorylate 10 mgs of protein. Since the calculation is based on assumption, additional experimental optimization may be needed.

Lysate Calculation Example

The first calculation is to convert the protein lysate sample from milligrams to molecules. This amount is important in determining the amount of each phosphatase needed. Since the lysate is a mixture of proteins an assumed average molecular weight of 100,000 daltons (g/mol) will be used to simplify the calculation. In this example there are 10 mgs of protein (2 mLs of lysate at 5 mg/mL) that will be treated with phosphatase enzyme.

Average Protein Molecular Weight: 100,000 g/mol

There are ~6.023 x 10¹⁶ molecules for 10 mgs of protein.

Lambda Phosphatase Calculation

The second calculation will be converting the number of lambda phosphatase units into molecules/minute. It is important to note that the reaction time and volume of enzyme can be adjusted once this has been determined.

For every 1 μl of lambda phosphatase enzyme added to the reaction, it will hydrolyze 2.41 x 10¹⁷ phosphate molecules. This final calculation can now determine how long the reaction will take.

It takes 15 seconds to completely dephosphorylate 10 mg of protein when using 1 μl of lambda phosphatase.

Alkaline Phosphatase Calculation

The same steps will be done to calculate the amount of alkaline phosphatase needed for the reaction. Notice the specific activity of Alkaline Phosphatase is different than Lambda Phosphatase.

It takes 0.045 seconds to completely dephosphorylate 10 mg of protein using 1 μl of alkaline phosphatase.

Other Helpful Links:

Interested in buying our lysis buffer? You can!

Following all these steps and still ending up with goop? Check out our blog on The Truth about Goopy Lysate!

Read even more about SDS as a lysis buffer in our blog: 1% SDS is the Lysis Buffer of Choice for Most Western Blots

1% SDS Lysis buffer

Help ensure your lysing success by purchasing our pre-made lysis buffer. With 1% SDS, it will completely solubilize membrane and other hard to solubilize proteins, and even synaptic junction proteins!