- Q. Can phosphorylated proteins be quantified？
A. They can be assayed on the basis of the band intensity by using a quantitative staining such as CBB staining. A product such as “Quick-CBB PLUS” is recommended.
⇒ Quick-CBB PLUS (1 L: Wako Cat. #178-00551; 250 mL: 174-00553)
- Q. Is there a limitation to the protein size for the application of Phos-tag™？
A. A phosphorylated protein of 350 kDa has actually been separated with 20 μM Phos-tag™, 3% acrylamide and 0.5% agarose*.
(See “[Ⅰ]-②’, ③’in “protocol” and “Tris-AcOH gel”.)
Reference: Proteomics, 9, 4098-4101 (2009), E. Kinoshita, E. Kinoshita-Kikuta, H. Uchijima, and K. Koike
＊ Agarose was added to strengthen the gel.
- Q. Is it possible to use gel-staining techniques other than CBB？
A. Yes, the gel can also be stained by negative staining, silver staining, and fluorescent staining.
Use of Phos-tag™ Acrylamide
- Q. How many gels can be made with each product？
A. It depends on the concentration of Phos-tag™ used.
For example, about 100 plates at 20 μM, about 40 plates at 50 μM, and about 20 plates at 100 μM can be prepared from a 10 mg-package, when gels of 1 mm-thickness,9 cm-width, and 7.7 cm-length are made.
|Phos-tag™||20 μM||50 μM||100 μM|
|0.3 mL Aqueous Soln. (0.9 mg )||abt. 9
|2 mg package||abt. 20
|10 mg package||abt. 100
- Q. The gel is too soft. What can I do for this？
A. Please refer to "Trouble Shooting.
Stability of the prepared gel containing Phos-tag™
- Q. How long can the prepared gel containing Phos-tag™ Acrylamide be stored？
A. Mn2+-Phos-tag™ SDS-PAGE gel cannot be stored. Use the gel on the day after preparation.
Zn2+-Phos-tag™ SDS-PAGE gel can be stored in a refrigerator for 3 months.
The gel deteriorates within a few days. Therefore, it should be prepared just before use.
Stability of the Phos-tag™ solution
- Q. How long can the solutions in methanol and water be stored？
A. No remarkable decline in performance has been reported for 1 year by refrigeration under protection from light.
Preparation of the reagent
- Q. Does the concentration of Phos-tag™ influence the amount of ions to be required？
A. The molar ratio of Phos-tag™ acrylamide to Mn2+ should be 1:2; two Mn2+ ions bind to one Phos-tag™ molecule (Fig. 1).
- Q. I have experienced clouding of Phos-tag™ when I prepared a solution as described in the protocol. Is this normal？
A. Yes, it is. Clouding is attributed to methanol. The solution becomes clear after standing for a while.
- Q. Does Phos-tag™ dissolve in water alone？
A. It is soluble in water, though it takes more time compared to dissolution in water containing methanol. If it does not dissolve completely, centrifuge the solution and use the supernatant.
- Q. What prestained markers can we use？
A. Using a prestained marker with the Phos-tag™ gel usually causes distortion of bands (Fig. 2). WIDE-VIEW™ Prestained Protein Size Marker Ⅲ (Wako Cat No. 230-02461) is less likely to cause band distortion, but does not reflect the molecular weight. Please use the result obtained using this marker as an index of the WB transfer efficiency. At least one blank lane is needed between the solution containing this marker and other solutions.
Phosphorylation reaction with coexisting ATP
- Q. Does ATP in a phosphorylation reaction solution affect electrophoresis？
A. ATP has no particular effect at a concentration of 2.0 mM. The limit of use has not been investigated yet.
- Q. Can we use Phos-tag™ Acrylamide in an ordinary precast gel by adding it to sample solution？
A. No, you cannot. We have various kinds of precast Phos-tag™ gels called “SuperSep Phos-tag™.
Interpretation of multiple bands
- Q. A band shift was observed in Phos-tag™ SDS-PAGE and multiple bands were detected. How can I decide whether the observation demonstrates phosphorylation or just degradation of the target protein？
A. Perform an ordinary SDS-PAGE (not using Phos-tag™) simultaneously to confirm that the target protein has not been degraded.
DNA Separation using Phos-tag™
- Q. Is Phos-tag™ applicable to separate DNA？
A. Refer to the following articles：
・A SNP genotyping method using phosphate-affinity polyacrylamide gel electrophoresis, Analytical Biochemistry, 361, 294-298 (2007), E. Kinoshita, E. Kinoshita-Kikuta, and T. Koike (The phosphate group at DNA-terminal is efficiently captured by Zn2+-Phos-tag™.)
・A mobility shift detection method for DNA methylation analysis using phosphate affinity polyacrylamide gel electrophoresis, Analytical Biochemistry, 378, 102-104 (2008), E. Kinoshita-Kikuta, E. Kinoshita, and T. Koike
- Q. What is the difference between BTL-104 and BTL-111？
A. BTL-104 and BTL-111 have linkers with different lengths.
BTL-104 is recommendable as the first choice because of its high solubility. BTL-111 offers high sensitivity.
- Q. What is the sensitivity level like？
A. It is at the nanogram level. Use a high-luminescence reagent such as ImmunoStar LD.
Other reagents required
- Q. Are there any reagents required other than the product？
A. Prepare a chemiluminescent reagent such as streptavidin-conjugated HRP solution and ImmunoStar Series. See "10" for ImmunoStar Series.
- Q. For how many tests can Phos-tag™ Biotin be used？
A. Please refer to the following as a guide.
BTL-104： 130〜1300 tests
BTL-111 1 mM Aqueous Solution： 10〜100 tests
- Q. Can phosphorylated proteins be assayed？
A. You can do semi-quantitative assay based on the density of bands.
- Q. Is it possible to determine the number of bound phosphate groups？
A. No, it isn't.
- Q. Is the product applicable to the staining of cells and tissues？
A. No. It is unsuitable for this application because it is removed by washing with methanol, etc.
- Q. Can I strip Phos-tag™ Biotin？
A. Yes, you can. Mix it with a solution containing 62.5 mM of Tris-HCl (pH 6.8), 2% (w/v) of SDS, and 0.1 M of 2-mercaptoethanol and shake the mixture for 15 minutes. Then, wash the mixture with 1 x TBS-T three times for 10 minutes each time. For further details, please contact us.
- Q. What kind of membrane is recommended？
A. We recommend PVDF membranes.
- Q. Does the use of Phos-tag™ Biotin require blocking？
A. No, it doesn't. Blocking causes the sensitivity to drop.
- Q. For how many tests can Phos-tag™ Mass Analytical kit be used？
A.More than 1,000 tests when 5 μL is used per test.
- Q. How can I know which one of Phos-tag™ MS-101L, Phos-tag™ MS-101H, and Phos-tag™ MS-101N is appropriate？
A. Phos-tag™ 101N contains naturally occurring zinc species, 101L contains 64Zn, and 101H contains 68Zn.
Please refer to the following guidance: ① For exploring the conditions: Use 101N.
② Many isotopes contained in it make the spectrum complicated. Verification of the presence of phosphate groups: Use 101L and 101H.
These reagents contain zinc with a mass number of 64 and 68, respectively.
Measurement of a single sample with these reagents therefore results in a difference in m/e of 8.
Detection of non-phosphorylated molecules
- Q. Why are the peaks of non-phosphorylated molecules not detected？
A. Because the ionization efficiency differs considerably between phosphorylated molecules and nonphosphorylated molecules. The sample solution using Phos-tag™ is suited to the method using a buffer of pH 6 to 8 and a weak-acidic phenol matrix (such as THAP) and weak-basic HAMAN.
In the peptide analysis in the general positive mode, on the other hand, acidic sample solutions and acidic matrix are used.
Therefore, the ionization efficiency of the phosphorylated molecule-Phos-tag™ complex increases dramatically whereas that of the non-phosphorylated molecules becomes extremely low.
- Q. I would like to measure a sample isolated by Phos-tag™ SDS-PAGE. Is it necessary to remove Phos-tag™ before in-gel digestion？
A. No, it isn't. Please follow the usual procedure for in-gel digestion after SDS-PAGE.
- Q. Can it be also used for ESI mass spectrometry？
A. Yes, it can. Please refer to the following publication, which reports an example of ESI-MS analysis in which Phos-tag™ MS-101N was used as probe. A neutral solution should be used because analysis in an acidic solution causes Phos-tag™ to be detached.
Reference: Anal. Chem. (2008), 80, 2531-2538 (MS-101N ESI-MS)
- Q. Can samples purified by using Phos-tag™ Agarose be directly applied to SDS-PAGE？
A. No, they can't. The elution buffer recommended in the protocol contains a high concentration of salt and may cause the bands to be distorted. Please use the SDS-PAGE sample buffer as elution buffer.
- Q. Is Phos-tag™ Agarose reusable？
A. We do not recommend it.
- Q. Does Phos-tag™ Agarose have any advantages over IMAC？
A. Phos-tag™ Agarose allows experimental processes under physiological conditions (pH 7.5), and since it does not use reductants or surfactants, it can refine phosphorylated proteins in their native shape.
Also, the purified proteins can be used in processes such as mass spectrometry and Western blotting.
Purification of His-tag proteins
- Q. Is the product applicable to purification of His-tag phosphorylated proteins？
A. As His-tag exhibits weak affinity to Zn2+, use a tag of other types such as GST, if possible.
It is assumed that Zn2+ shows a higher affinity to Phos-tag™ than to His-tag since isolation of His-tag protein by Zn2+ -Phos-tag™ SDS-PAGE has been reported although no result of purification of His-tag protein with Phos-tag™ Agarose has been confirmed.
- Q. What reagents are suitable and unsuitable for use in the sample preparation？
A. Please refer to the table below.
|Reducing agents||DTT||○||≤ 0.1 M|
|Denaturing agents||Urea||○||Using it at 8M has no negative effect.|
|SDS||○||Using it at ≥ 0.5% affects the binding process.|
|Sodium deoxycholate||○||Using it at ≥ 0.25% affects the binding process.|
|Nonidet P40||○||≤ 1 %|
|Tween 20||○||≤ 1 %|
|CHAPS||○||≤ 0.2 %|
|Phosphate derivatives||β-Glycerophosphate||x||Do not use it.|
|Pyrophosphate||x||Do not use it.|
|Chelating agents||EDTA||x||Do not use it.|