- Q. Can phosphorylated proteins be assayed？
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. How large (kDa) can a protein be separated using this product？
A. phosphorylated protein of 350 kDa has actually been separated with 20 μM Phos-tag™, 3% acrylamide and 0.5% agarose＊.
Reference: Proteomics, 9, 4098-4101 (2009), E. Kinoshita, E. Kinoshita-Kikuta, H. Uchijima, and K. Koike
＊ Agarose was added to strengthen the gel.
- Q. How can the resolution be improved？
A. In general, a higher concentration of Phos-tag™ results in a higher resolution. However, increasing the concentration of Phos-tag™ also causes the overall migration speed of the protein to proportionally drop. Please refer to the page.
- 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||abt. 4||abt. 2|
|2 mg package||abt. 20||abt. 8||abt. 4|
|10 mg package||abt. 100||abt. 40||abt. 20|
|SuperSep Phos-tag™||-||5 gels||-|
- Q. The gel is easily broken. What can I do for this？
A. A low concentration of acrylamide causes the gel to be soft.
You can solve this problem by increasing the relative amount of methylenebisacrylamide to acrylamide (24:1), for example.
Stability of the prepared gel containing Phos-tag™
- Q. How long can the prepared gel containing Phos-tag™ Acrylamide be stored？
A. 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 6 months by refrigeration under protection from light. The solutions seem to be storable for 1 year without any problem according to doctors who are using the product.
Phos-tag™ Acrylamide (continued)
Preparation of the reagent
- Q. Does the concentration of Phos-tag™ influence the amount of ions required to be
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 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 had 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 a precast gel by adding it to sample solution？
A. No, you cannot. We have various kinds of precast gels called “SuperSep Phos-tag™ shown on the page.
Differentiation between degraded protein and phosphorylated Phos-tag™
- Q. A mobility shift was observed in Phos-tag™ SDS-PAGE. How can I know whether it is a sign of phosphorylation or only telling me that the protein was broken down？
A. Please carry out a conventional SDS-PAGE (without Phos-tag™) and verify that your protein is intact.
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
SuperSep Phos-tag™（Please see the page.）
- Q. Do you have lower-concentration polyacrylamide products？
A. Products with concentrations of 6%, 7.5% and 10% are currently being developed.
- Q. Do you have products with other Phos-tag™ polyacrylamide concentrations？
A. Products with concentrations of 20 μM and 100 μM are currently being developed.
- Q. Are there ways to improve separation capacity？
A. Using a Tris-Tricine buffer as the running buffer improves separation capacity.
- Q. Are there any references？
A. Kinoshita-Kikuta, E., Kinoshita, E., Koike T., "A Laborsaving, Timesaving, and More Reliable Strategy for Separation of Low-Molecular-Mass Phosphoproteins in Phos-tag Affinity Electrophoresis", Int. J. Chem. 4, 1-8 (2012) DOI: 10.5539/ijc.v4n5p1.
Phos-tag™ Biotin（Please see the page.）
- Q. What is the difference of BTL-104, BTL-105 and BTL-111？
A. BTL-104, BTL-105, and BTL-111 have linkers with different lengths. Although the usage of BTL-104 and BTL-105 are similar, 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.
- Q. Do we need other reagents besides this product？
A. Prepare a Streptavidin-conjugated HRP solution.
- Q. How many times can Phos-tag™ Biotin be used？
A. It depends on the frequency of use. Please refer to the following as a guide.
BTL-104： 130〜1300 times
BTL-105： 113〜1130 times
BTL-111 1 mM Aqueous Solution： 10〜100 times
- 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 binding phosphate groups？
A. No, it isn't.
- Q. Can I strip the antibodies of 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×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.
Phos-tag™ Mass Analytical Kit（Please see the page.）
- Q. How many tests can Phos-tag™ Biotin 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. Exploration of conditions: Use 101N.
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 16.
- 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)
Phos-tag™ Agarose（Please see the page.）
- Q. Can samples purified 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 all 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.
- 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||×||Do not use it.|
|Pyrophosphate||×||Do not use it.|
|Chelating agents||EDTA||△||Using it at a high concentration has a negative effect.|