本产品专为 Phos-key SDS-PAGE 系统设计，适用于磷酸化蛋白样品的上样与分离。采用中性配方，可在 pH 5–8 范围内与 Phos-key 形成稳定的复合物，内含 SDS 及 β-巯基乙醇。
Phos-key 技术原理：
Phos-key 是一种能与磷酸基团特异性结合的双核金属络合物。在常规 SDS-PAGE 凝胶中掺入 Phos-key 及二价金属离子（Mn²⁺ 或 Zn²⁺），电泳时凝胶内的 Phos-key 复合物可特异性结合磷酸化蛋白，使其电泳迁移率降低，从而实现磷酸化与非磷酸化蛋白的有效分离。
产品优势：
• 摆脱抗体依赖：可检测不同形式的磷酸化蛋白，不受特异性磷酸化抗体限制。
• 下游兼容性强：分离后的蛋白条带可直接用于免疫印迹（Western Blot）及质谱分析。
实验步骤（仅供参考）：
1. 按照每 2 μL 蛋白样品加入 1 μL 上样缓冲液(3×)的比例混合；
2. 混匀后，95℃孵育 5 - 10 min，以充分变性蛋白；
3. 冷却至室温后，用微量移液枪上样到 PAGE 胶加样孔内；
4. 当溴酚蓝到达 PAGE 胶底部附近时即可停止电泳。
Phos-key SDS-PAGE 样品制备的要求：
1．确保样本中不含有 EDTA 等金属离子螯合剂;
2．样品中含有的还原剂、变性剂、螯合剂、钒酸等会使电泳条带发生弯曲或者拖尾，可通过 TCA 沉淀或透析脱盐降低杂质含量；
3．避免样品中含有高浓度的还原剂，变性剂，表面活性剂等；β-巯基乙醇浓度不高于 0.2 M（或者 5%）；
4．ATP 使用浓度在 2.0 mM 时，对实验结果无显著影响，具体使用范围尚不清楚；
5．样品可以为纯化蛋白、组织均浆液、组织裂解液、体外酶反应液，且样品来源无物种特异性。
注意事项：
1. 本产品为 3× 母液。
2. 建议分装冻存，避免反复冻融。
3. 仅限于专业人员科学研究用，不得用于临床医学诊断及其他非合理用途。不得存放于普通住宅内。
4. 为了您的安全和健康，请穿戴好个人防护装备和实验服装进行操作。
This product is designed exclusively for the Phos-key SDS-PAGE system for loading and separation of phosphoprotein samples. It adopts a neutral formulation and can form stable complexes with Phos-key within the pH range of 5–8. The buffer contains SDS and β-mercaptoethanol.
Phos-key Technical Principle:
Phos-key is a dinuclear metal complex that binds specifically to phosphate groups. When Phos-key and divalent metal ions (Mn²⁺ or Zn²⁺) are incorporated into conventional SDS-PAGE gels, the Phos-key complexes within the gel specifically bind phosphoproteins during electrophoresis. This reduces the electrophoretic mobility of phosphoproteins, enabling effective separation of phosphorylated and non-phosphorylated proteins.
Product Advantages:
• Antibody-independent detection: Detects various forms of phosphoproteins without reliance on phospho-specific antibodies.
• Broad downstream compatibility: Resolved protein bands are directly compatible with Western blot and mass spectrometry analysis.
Procedure (for reference only):
1.Mix at a ratio of 1 μL of loading buffer (3×) per 2 μL of protein sample.
2.After mixing, incubate at 95°C for 5–10 min to fully denature the proteins.
3.Cool to room temperature, then load into the wells of the PAGE gel using a micropipette.
4.Stop electrophoresis when the bromophenol blue reaches near the bottom of the PAGE gel.
Requirements for Phos-key SDS-PAGE Sample Preparation:
1.Ensure that the sample does not contain metal ion chelators such as EDTA.
2.Reducing agents, denaturants, chelators, vanadate, etc., present in the sample may cause band distortion or smearing during electrophoresis. Impurity levels can be reduced by TCA precipitation or dialysis/desalting.
3.Avoid high concentrations of reducing agents, denaturants, surfactants, etc., in the sample; the concentration of β-mercaptoethanol should not exceed 0.2 M (or 5%).
4.When ATP is used at a concentration of 2.0 mM, it has no significant effect on the experimental results; the specific usable range remains unclear.
5.The sample can be purified protein, tissue homogenate, tissue lysate, or in vitro enzyme reaction mixture, and the sample source is not species-specific.
Precautions:
1.This product is a 3× stock solution.
2.It is recommended to aliquot and freeze for storage to avoid repeated freeze-thaw cycles.
3.For research use by professionals only. It must not be used for clinical medical diagnosis or other unreasonable purposes. It must not be stored in ordinary residential buildings.
4.For your safety and health, please wear appropriate personal protective equipment and lab clothing during operation.
危险品化学品经营许可证（带存储）