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磷酸鹽緩沖液需要稀釋到多少才可以使用？

更新時間：2025-08-29  |  點擊率：78

磷酸鹽緩沖液(PBS)是否需要稀釋以及稀釋到何種濃度，主要取決于具體實驗需求。以下是不同場景下的稀釋建議及注意事項：

一、是否需要稀釋？

  1. 無需稀釋的場景
    • 細(xì)胞實驗：標(biāo)準(zhǔn)PBS(0.01 M，pH 7.4)可直接用于細(xì)胞洗滌、重懸或傳代培養(yǎng)。
    • 分子生物學(xué)實驗：如Western blot、ELISA中樣本稀釋或洗滌，通常使用標(biāo)準(zhǔn)濃度。
    • 組織學(xué)處理：組織切片漂洗、抗原修復(fù)后洗滌等，標(biāo)準(zhǔn)濃度即可滿足需求。
  2. 需要稀釋的場景
    • 高鹽敏感實驗：如某些蛋白質(zhì)透析或酶活性測定，需降低NaCl濃度(如稀釋至0.14 M)。
    • 特殊細(xì)胞類型：某些原代細(xì)胞(如神經(jīng)元)對滲透壓敏感，需用低鹽PBS(如0.001 M磷酸鹽+0.137 M NaCl)。
    • 藥物穩(wěn)定性測試：模擬體內(nèi)低鹽環(huán)境時，可能需稀釋至生理鹽水濃度(0.9% NaCl，約0.15 M)。

二、常見稀釋比例及用途

稀釋比例 目標(biāo)濃度 典型用途
1:1（原液） 0.01 M磷酸鹽 + 0.137 M NaCl 細(xì)胞培養(yǎng)、Western blot、ELISA、免疫組化洗滌
1:10 0.001 M磷酸鹽 + 0.0137 M NaCl 特殊細(xì)胞（如神經(jīng)元）洗滌、低鹽需求實驗
1:100 0.0001 M磷酸鹽 + 0.00137 M NaCl **低鹽環(huán)境模擬（如某些酶活性測定）
調(diào)整NaCl濃度 0.01 M磷酸鹽 + 0.014 M NaCl 降低滲透壓至生理鹽水水平（0.9% NaCl）

三、稀釋方法

  1. 體積比稀釋
    • 示例：將10 mL標(biāo)準(zhǔn)PBS加入90 mL去離子水，得到1:10稀釋液。
    • 工具：使用移液器或量筒精確量取，避免污染。
  2. 質(zhì)量體積比稀釋
    • 適用場景：需精確控制NaCl濃度時（如配制0.01 M磷酸鹽 + 0.014 M NaCl）。
    • 計算：根據(jù)目標(biāo)濃度計算所需NaCl質(zhì)量（如0.014 M × 58.44 g/mol × 1 L = 0.818 g NaCl/L）。
  3. 分步稀釋
    • 步驟：先稀釋磷酸鹽濃度，再調(diào)整NaCl濃度（如需同時降低兩者）。

四、稀釋后驗證

  1. pH測量
    • 稀釋可能改變pH，需用pH計重新測定并調(diào)節(jié)至目標(biāo)值（如7.4）。
    • 調(diào)整方法：滴加濃鹽酸（降低pH）或氫氧化鈉（升高pH）。
  2. 滲透壓檢測
    • 使用滲透壓計驗證稀釋液是否符合實驗要求（如細(xì)胞培養(yǎng)需280-320 mOsm/kg）。
  3. 無菌性檢查
    • 若稀釋后用于細(xì)胞實驗，需通過濾膜過濾（0.22 μm）或高壓滅菌確保無菌。

五、注意事項

  1. 避免反復(fù)凍融
    • 稀釋后的PBS應(yīng)分裝保存，避免反復(fù)解凍導(dǎo)致pH或滲透壓變化。
  2. 兼容性測試
    • *次使用稀釋液時，需進(jìn)行預(yù)實驗驗證其對細(xì)胞活性或?qū)嶒灲Y(jié)果的影響（如MTT實驗檢測細(xì)胞毒性）。
  3. 標(biāo)記與記錄
    • 稀釋液需明確標(biāo)注濃度、pH、配制日期及滅菌狀態(tài)，避免混淆。
  4. 特殊實驗需求
    • 透皮吸收實驗：可能需稀釋至與接收液成分一致（如含4% BSA的PBS）。
    • 蛋白質(zhì)電泳：需使用無NaCl的磷酸鹽緩沖液（如Tris-Glycine系統(tǒng)）。
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