SuperSep™ Phos-tag^™ Precast Gels

Albumin Dephosphorylation

Time-dependent Dephosphorylation of β-casein

[Electrophoresis Buffer] Tris-Glycine SDS buffer
[Electrophoresis Samples]
　Lane1: β-casein (AP-treatment: 0 min)
　Lane2: β-casein (AP-treatment: 15 min)
　Lane3: β-casein (AP-treatment: 30 min)
　Lane4: β-casein (AP-treatment: 45 min)
　Lane5: β-casein (AP-treatment: 60 min)
[Electrophoresis Conditions] 35mA, 60min
[Staining] Quick CBB staining
[Destaining] Deionized Water

Time-dependent Change in the level of phosphorylation

Procedure

X-ray irradiation (5 Gy) was performed for human lung cancer cell line Lu99, and cells were collected across different time points. Cell extracts were prepared, and SDS-PAGE was performed in 13 wells using 50 μmol/L of 10% SuperSep™ Phos-tag™

Gentle agitation of the gel was performed in a transfer buffer containing 10 mmol/L EDTA, and proteins were transferred to PVDF membrane. The membrane was blocked with 2% Milk/TBS-T, and was reacted with primary antibodies (upper image: p53, lower image: cell-cycle proteins). Detection was performed by chemoluminecence.

Results

Protein accumulation for p53 was at the highest level 4 hours after X-ray irradiation. The level of protein X phosphorylation changed over time after X-ray irradiation.

Electrophoresis of Control Protein

Electrophoresis Conditions

30 mA/gel (constant), 60 mins

Sample

5 μg/lane of α-casein from bovine milk, dephosphorylated (Cat. No. 038-23221)

• The product contains α-casein that hasve not been dephosphorylated. While conventional SDS-PAGE will only show a single main band, Phos-tag^™ SDS-PAGE will show two main bands for α-casein and dephosphorylated α-casein.

Running Buffer

Staining

Quick CBB Plus (Cat. No. 174-00553)

For Bio-Rad Mini-PROTEAN^® Tank

198-17981	SuperSep Phos-tag (50μmol/l), 7.5%, 17well, 83×100×3.9 mm
195-17991	SuperSep Phos-tag (50μmol/l), 12.5%, 17well, 83×100×3.9 mm

For Invitrogen^® XCell SureLock Mini-Cell Tank

192-18001	SuperSep Phos-tag (50μmol/l), 7.5%, 17well, 100×100×6.6 mm
199-18011	SuperSep Phos-tag (50μmol/l), 12.5%, 17well, 100×100×6.6 mm

For FUJIFILM Wako Easy Separator™

192-17401	SuperSep Phos-tag (50μmol/l), 6%, 13well
199-17391	SuperSep Phos-tag (50μmol/l), 6%, 17well
193-16711	SuperSep Phos-tag (50μmol/l), 10%, 13well
190-16721	SuperSep Phos-tag (50μmol/l), 10%, 17well
195-17371	SuperSep Phos-tag (50μmol/l), 7.5%, 13well
192-17381	SuperSep Phos-tag (50μmol/l), 7.5%, 17well
195-16391	SuperSep Phos-tag (50μmol/l), 12.5%, 13well
193-16571	SuperSep Phos-tag (50μmol/l), 12.5%, 17well
193-16691	SuperSep Phos-tag (50μmol/l), 15%, 13well
196-16701	SuperSep Phos-tag (50μmol/l), 15%, 17well

Q1. What electrophoresis tanks are the Phos-tagTM Precast Gels compatible with?

Phos-tag Precast Gels are available in three cassette sizes, [83 x 100 x 3.9 mm], [100 x 100 x 3.0 mm] and [100 x 100 x 6.6 mm].

• [83 x 100 x 3.9 mm] is suitable for Bio-Rad Mini-PROTEAN Tank.
• [100 x 100 x 3.0 mm] is suitable for Wako Easy Separator.
• [100 x 100 x 6.6 mm] is suitable for Invitrogen^® XCell SureLock^® Mini-Cell Tank.

Q2. What gel staining methods can we use?

CBB, negative, silver, and florescent staining methods can be used.

Q3. CBB-stained gel cannot be de-stained clearly.

A microwave can be used when de-staining to give you a clearer gel.

Procedure

Transfer the stained gel in 100 mL of deionized water in the container, add a few rolls of Kim-Wipes, and heat it in a microwave for a few minutes. Replace deionized water and repeat the same procedure 2-3 times. Be careful with handling as the container will be hot.

Q4. Can the product be used for Western blot?

Yes: However, zinc in the gel has to be removed by EDTA treatment to achieve optimal transfer efficiency to the membrane.

Procedure

Immerse the gel in a transfer buffer (25 mmol/L Tris, 192 mmol/L Glycine, 10% MeOH) containing 10 mmol/L EDTA, and agitate gently for 10 minutes. Repeat the procedure 2 times. Replace with a transfer buffer (25 mmol/L Tris, 192 mmol/L Glycine, 10% MeOH) that does not contain EDTA, agitate for 10 minutes, and transfer onto PVDF membrane or nitrocellulose membrane. If the transfer efficiency is poor, consider increasing the frequency of EDTA treatment or the concentration of EDTA.

Q5. Bands are distorted.

Distortion and tailing of bands can be observed with samples that contain EDTA, inorganic salts, and detergents. In such cases, it is effective to desalt samples by dialysis or precipitation with TCA. The presence of an empty lane can also cause band distortion. Apply a sample buffer (1x) in empty lanes to avoid distortion.

Q6. Phosphorylated and dephosphorylated target proteins are not separated.

Perform electrophoresis and confirm band shift with β-casein (positive control) and alkaline phosphatase-treated β-casein (negative control). If there is a band shift, it is likely that phosphorylated and dephosphorylated variants of your target protein are not separated with the Phos-tag^™ and/or acrylamide concentrations of this product.

Q7. Can we use crude extracts of cells with this product?

Yes: However, the use of proteins with small Rf values may result in unclear bands.

Q8. What is the appropriate amount of a sample that should be applied to a well?

1-5 µg for purified proteins with CBB staining, and 10-30 µg for tissue or cell extracts (adjust the amount depending on the level of protein expression).

• These are only recommended amounts. SDS-PAGE or Western blot should be performed to determine the appropriate amount of a sample.

Q9. What molecular markers should we use?

There are no specific markers that we recommend as the product is not influenced by the molecular weight of a marker. Instead of a marker, E. coli -derived recombination proteins or dephosphorylated samples are recommended as negative controls.

Q10. Which ion complex is contained in the product?

The product contains zinc ion.

Q11. How can we determine whether the band shift is caused by phosphorylation?

Perform gel electrophoresis with 12.5% SuperSep™ Ace (Cat. No. 199-14971) with the same gel concentration to confirm that the target protein is not degraded.