This plan only provides a guide, please modify it to meet your specific needs.
1、Solutions for Phos-tag Biotin BTL-104 Method
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Sol. A ：Tris buffered saline
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Tris buffered saline (10xTBS, 1 L, pH 7.5)
Tris (0.10 mol/L) -----------------------------------------------12.1 g
NaCl (1.0 mol/L) -----------------------------------------------58.4 g
distilled water-----------------------------------------------------0.9 L
2 mol/L aqueous HCl for pH adjustment at 7.5--------------a proper quantity
distilled water for preparation of the 1 L solution-----------a proper quantity
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Sol. B: Tween 20 solution
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10% (v/v) Tween 20 solution (50 mL)
Tween 20 ----------------------------------------------------------5 mL
distilled water ---------------------------------------------------45 mL
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Sol. C: TBS-T
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1xTBS-T (1 L)
Sol. A ----------------------------------------------------------100 mL
Sol. B -----------------------------------------------------------10 mL
distilled water ------------------------------------------------890 mL
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Sol. D: Phos-tag Biotin BTL-104 methanol solution
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Phos-tag Biotin BTL-104 methanol solutions
Phos-tag Biotin BTL-104 (MW: 767)--------------------------10 mg
Methanol-----------------------------------------------0.13 mL
【Storage】Stored in a dark place at 4℃.
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Sol. E: Phos-tag Biotin BTL-104 solution
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Phos-tag Biotin BTL-104 solution (10 mmol/L)
Sol. D-------------------------------------------------------0.13 mL
Sol. C-------------------------------------------------------1.17 mL
【Storage】Stored in a dark place at 4℃.
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Sol. F: ZnCl2 aqueous solution
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10 mmol/L ZnCl2 aqueous solution (50 mL)
ZnCl2 (FW. 136.3)---------------------------------- 68.15 mg
Distilled water --------------------------------------------------50 mL
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Sol. G: Streptavidin-conjugated Horseradish Peroxidase solution
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Streptavidin-Horseradish Peroxidase Conjugate
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2、Preparation of Phos-tag Biotin-Streptavidin-conjugated HRP
1) After mixing of the following solutions, the obtained solution (Sol. H) is allowed to stand for 30 min at room temperature.
Sol. C (1xTBS-T) ------------------------------------------------------------------469 μL
Sol. E (Phos-tag Biotin BTL-104 solution) *------------------------------------------ 1 ~ 10 μL
Sol. F (10 mmol/L ZnCl2)------------------------------------------------ 2 ~ 20 μL
Sol. G (Streptavidin-conjugated Horseradish Peroxidase) --------------------- 1 μL
2) Sol. H is added in a centrifugal filter device cup (NMWL = 30,000, NanosepTM 30K). Seal with the attached cap.
3) Centrifuge (14,000 x g) for 20 min at room temperature to remove the excess Phos-tag Biotin BTL-104.
4) The remaining solution (<10 μL) in the cup is diluted with 30 mL of Sol. C (1xTBS-T), which is Sol. Phos-tag Biotin-SH (a solution of Zn2+-Phos-tag Biotin-bound Streptavidin-conjugated HRP**).
*: [Phos-tag Biotin BTL-104] >> [Streptavidin-conjugated HRP]
: Phos-tag Biotin-bound Streptavidin-conjugated HRP in Sol. Phos-tag Biotin-SH is stable for 30 days at 4 °C
3、Probing with Phos-tag Biotin-bound Streptavidin-conjugated HRP
1) A protein-blotted PVDF membrane is soaked with Sol. C (1xTBS-T) in a Tupperware. Use plastic gloves in this procedure. The membrane is gently rocked for at least 1 h. Confirm that the membrane does not stick to the Tupperware. Be careful not to dry the membrane.
2) The membrane is incubated with Sol. Phos-tag Biotin-SH (ca. 1 mL/5 cm2) in a plastic bag. The bag is gently rocked for 30 min.
3) The membrane is taken out of the bag and washed twice with Sol. C (ca. 10 mL/5 cm2) in a Tupperware for 5 min each time at room temperature (the Tupperware is gently rocked). Confirm that the membrane does not stick to the Tupperware. Be careful not to dry the membrane.
4) The chemiluminescence is observed using an X-ray film or an image analyzer with an appropriate amount of a chemiluminescence reagent (e.g., Ultra High Sensitivity ECL Kit GK10008).
4、Reprobing the Protein-blotted PVDF Membrane
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Sol. K: Tris-HCl buffer
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0.5 mol/L Tris-HCl buffer (1 L, pH 6.8)
Tris ----------------------------------------------------------- 60.6 g
distilled water--------------------------------------------------0.8 L
6 mol/L aqueous HCl for pH adjustment at 6.8----------a proper quantity
distilled water for preparation of the 1 L solution-------a proper quantity
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Sol. L: SDS solution
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10% (w/v) aqueous SDS solution (1 L)
SDS ----------------------------------------------------------100 g
distilled water for preparation of the 1 L solution-------a proper quantity
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Sol. M: Stripping buffer
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Stripping buffer (1 L)
Sol. K --------------------------------------------------------125 mL
Sol. L -------------------------------------------------------200 mL
2-mercaptoethanol ----------------------------------------- 7 mL
distilled water---------------------------------------------668 mL
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1) A protein-blotted PVDF membrane probed with Zn2+-Phos-tag Biotin and Streptavidin-conjugated HRP is soaked with Sol. C (1xTBS-T, 25 mL/5 cm2) in a Tupperware.
The membrane is gently rocked for at least 1 h. Be careful not to dry the membrane. If the membrane is dry, the membrane is soaked with methanol before this stripping treatment.
2) The membrane is soaked with Sol. M (Stripping buffer, 25 mL/5 cm2) in a Tupperware. The membrane is gently rocked for 20 min at room temperature. There is no need of heating. Confirm that the membrane does not stick to the Tupperware.
3) The membrane is soaked with Sol. C (1xTBS-T, 25 mL/5 cm2) in a Tupperware. The membrane is gently rocked for 1 h at room temperature. Wash 3 times. Confirm that the membrane does not stick to the Tupperware and be careful not to dry the membrane.
4) The membrane is blocked with an appropriate protein and then reprobed with an appropriate antibody. The chemiluminescent analysis is conducted.
5、Trouble Shooting
1) Phos-tag Biotin in Sol. E (10 μL) is large excess amount against Streptavidin-conjugated Horseradish Peroxidase in Sol. G (1 μL). We obtained almost the same result using smaller amount of Phos-tag Biotin (e.g., 1 μL Sol. E) and Sol. G (1 μL) used. The user should adjust the volume of Sol. D to obtain the required sensitivity or save expenses. If the volume of Sol. E is decreased, it is no need to change the volume of the zinc(II) solution (Sol. F).
2) If the membrane is not thoroughly soaked with Sol. C, the background signal is high and spotted. Furthermore, the protein signals are not observed (i.e., white spots in the right-side figure). Confirm that the membrane does not repel Sol. C.
3) PVDF membrane is highly recommended for the Phos-tag Biotin method.
6、FAQ
1) Q: What is the sensitivity level like?
A: It is at the nanogram level. Use a high-luminescence reagent such as ImmunoStar LD.
2) Q: Do we need other reagents besides this product?
A: Prepare a Streptavidin-conjugated HRP solution.
3) Q: How many times can Phos-tag Biotin BTL-104 be used?
A: It depends on the frequency of use. Please refer to the following as a guide. BTL-104: 130～1300 times
4) Q: Can phosphorylated proteins be assayed?
A: You can do semi-quantitative assay based on the density of bands.
5) Q: Is it possible to determine the number of binding phosphate groups?
A: No, it isn't.
6) Q: Can I strip the antibodies of Phos-tag Biotin BTL-104?
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.
7) Q: What kind of membrane is recommended?
A: We recommend PVDF membranes.
8) Q: Does the use of Phos-tag Biotin BTL-104 require blocking?
A: No, it doesn't. Blocking causes the sensitivity to drop.
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