Question Detail
What is the lower limit of linearity for the BCA kit for low-concentration proteins in fish serum?
Lab Techniques
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All Answers4
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Elisa2682
2026-07-01
动物样本我一般更推荐BCA，尤其裂解液含Triton、SDS时比Bradford稳，受蛋白氨基酸组成影响也更小。Bradford虽然快，但线性范围窄、对裂解液和脂肪更敏感，对鱼血清这种低浓度、背景高的样本容易波动。如果只是粗筛，可以Bradford；正式定量、Western上样用BCA。最好两法平行做一批，差异小再把BCA作为日常方法。
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Andy8162
2026-09-24
鱼血清蛋白再低，大多数仍高于0.05 mg/mL，要注意稀释比例。若确实很低，建议减少稀释或先离心超滤浓缩。低浓度时背景影响大，标准品稀释液要尽量与样品缓冲液一致，并设样品空白。复孔至少3个，看板内CV；如果CV超过10%，不要硬读，考虑换高灵敏BCA或改变上样量。
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ProteinTech4173
2026-09-11
常规BCA的线性低端标称一般在20-25 µg/mL左右，但0.05 mg/mL已经接近低端，想测准要看标曲和空白。建议标准曲线设置0、0.025、0.05、0.1、0.2、0.5、1.0、2.0 mg/mL，采用二次或四参数拟合；样品可增加上样量至25ul或采用60℃ 30min增强法提高吸光度。若0.05 mg/mL样品与空白的吸光度差值小于0.03-0.05，重复性会下降。
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SamAntibody5983
2026-08-30
鱼肌肉脂肪高，匀浆后要高速离心去脂。可在4℃ 12000g离心10-15min，吸去上层白色脂层，取中间清液。若样品仍浑浊，用0.45um低温滤器过滤或冷冻离心。脂肪残留会使孔内出现分相、气泡和光散射，影响酶标仪读数。匀浆时用含1% Triton的裂解液有助于溶解和释放蛋白，但标准曲线必须同buffer。
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