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细菌蛋白抽提试剂
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为什么选择此级别
BioReagent,细菌抽提用 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
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2-8°C储存。低温运输 。请查阅批次 COA 获取详细规格。
质量文档
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
细菌蛋白提取试剂，又称细菌活性蛋白提取试剂，是一款用于分离活性细菌蛋白的裂解缓冲液。它能够实现快速、高质量和高产量的直接裂解和蛋白质提取，无需超声波处理或高压均质破碎。该试剂适用于大肠杆菌内源性可溶性蛋白和在大肠杆菌中表达的重组蛋白，非常适合高通量快速蛋白质表达筛选。
该试剂适用于新鲜制备的细菌细胞和冷冻细胞沉淀物，冷冻样本中的提取性能尤为出色。所需剂量由细胞沉淀物的重量决定。不含 EDTA 的蛋白酶抑制剂、盐类和还原剂可以直接添加到试剂系统中。无需添加外源酶即可实现高效的蛋白质提取。这种试剂能够提取可溶性蛋白质，并可用于去除包涵体表面的碎屑和污染物，从而获得高纯度的包涵体蛋白质。需要注意的是，不能溶解包涵体，可以洗涤包涵体以获得高纯度包涵体。可用于多种生物化学和分子生物学用途，兼容常见下游实验操作如蛋白的纯化(His标签蛋白建议选用Ni-TED，若选用Ni-IDA或Ni-NTA，以及GST和MBP标签蛋白的纯化，建议稀释10倍或透析后可进行)、ELISA、Western blot等。
使用方法
1. 小量可溶性蛋白抽提。通过小量蛋白抽提实验来检测目的蛋白是否表达和表达量，然后再进行大量的蛋白提取。
1.1 按照常规方法进行重组蛋白表达菌株的培养以及目的蛋白的诱导表达。
1.2 取1.5ml OD600为0.5-2.0的菌液，12,000-16,000g 4℃或室温离心2min，弃上清。
1.3 用0.2-0.4ml 细菌蛋白抽提试剂重悬细胞，可短暂涡旋混合以充分重悬。室温孵育1min以上。
1.4 12,000-16,000g (16,000g更佳) 4℃离心5min。
1.5 小心吸取上清，即为抽提获得的可溶性蛋白。吸取上清时触及沉淀会导致抽提产物中含有更多的杂蛋白等。
1.6 通过SDS-PAGE或Western blot等方法检测目的蛋白存在于上清还是不溶性沉淀中。SDS-PAGE电泳时，每个样品的上样量推荐为5-15μl。
2. 大量可溶性蛋白抽提。
2.1 按照常规方法进行重组蛋白表达菌株的培养以及目的蛋白的诱导表达。
2.2 收集250ml OD600约为2.0的菌液，5,000g 4℃或室温离心10min，弃上清后可获得约1g的湿菌。后续可以直接用于裂解或冻存后用于裂解，通常冻存后再裂解可以获得更高的蛋白得率。但冻融对于某些蛋白的活性可能会起到负面影响。
2.3 按照每1g湿菌加入20-50ml 细菌活性蛋白抽提试剂，在使用前可加入适当的蛋白酶抑制剂的比例进行抽提，用移液管反复吹打混匀。加入较少的抽提试剂可以获得较高浓度的蛋白，但抽提效率会有所下降；加入较多的抽提试剂可以获得更高的抽提效率，但蛋白浓度会相对较低。如果较易取得比较多的湿菌，推荐使用较少的抽提试剂。此外，为取得最佳结果，可以加入最终浓度为50U/ml的核酸酶或重组全能核酸酶以降低粘稠度。
2.4 室温孵育1min以上，可适当延长时间以充分抽提蛋白。
2.5 12,000-16,000g (16,000g更佳) 4℃离心10min。
2.6 小心吸取上清，即为抽提获得的可溶性蛋白。吸取上清时触及沉淀会导致抽提产物中含有更多的杂蛋白等。
2.7 通过SDS-PAGE或Western blot等方法检测目的蛋白存在于上清还是不溶性沉淀中。SDS-PAGE电泳时，每个样品的上样量推荐为5-15μl。
常见问题
1. 目的蛋白产量低。
(1) 细菌裂解不充分。反复冻融细菌促进细胞破裂，或者加入溶菌酶都有助于蛋白的抽提。
(2) 样品的粘稠度太高。加入核酸酶或重组全能核酸酶可降低样品的粘稠度，有助于可溶性蛋白的抽提。
(3) 目的蛋白降解了。加入蛋白酶抑制剂可降低目的蛋白的降解。
(4) 目的蛋白的表达水平低。可加入较高浓度的IPTG、延长诱导时间、调整诱导温度等，同时考虑检查构建的质粒，或选用其它蛋白表达菌株。
(5) 目的蛋白可能是不溶性的。检查离心后的沉淀，确定目的蛋白是否形成了包涵体。
(6) 蛋白抽提试剂加入量太少。适量增加蛋白抽提试剂的量。
2. 抽提的可溶性蛋白溶液浑浊不透明。
(1) 加入的蛋白抽提试剂的量不足。适当增加抽提试剂的用量。
(2) 细菌裂解不充分。可以尝试在蛋白抽提的时候添加溶菌酶，添加溶菌酶后很可能会使最终的蛋白溶液变澄清。
(3) 抽提后的蛋白样品冻存时间过长。建议在1-2周内使用抽提后的蛋白溶液进行后续实验。
(4) 分离蛋白上清和沉淀时的离心力或离心时间不够。确保12,000-16,000g (16,000g更佳) 4℃离心10min ，或使用更大的离心力或离心更长时间。
(5) 表达的目的蛋白发生聚集。加入甘油至终浓度为40-50%通常可以阻止蛋白聚集和析出，或者用硫酸铵沉淀法将目的蛋白从抽提试剂中沉淀下来，再设法溶解。
(6) 低温导致蛋白溶解性下降。恢复到室温，增加蛋白溶解性可能会使溶液变澄清。这种情况下增加抽提试剂用量也可以解决该问题的。
3. 抽提的可溶性蛋白溶液呈粘稠状。
(1) 加入的蛋白抽提试剂的量不足。适当增加抽提试剂的用量。
(2) 加入核酸酶或重组全能核酸酶可降低样品的粘稠度。
注意事项
1. 可以考虑选购蛋白酶抑制剂混合物(P776695、P665818)或PMSF (100mM) (P301906)，并添加到本产品中以抑制蛋白降解。
2. 可以考虑选购重组全能核酸酶(O775185、rp221814)或核酸酶(B401536)，并添加到本产品中以降低抽提产物的粘稠度。
3. 本产品采用Tris-HCl缓冲系统，蛋白提取后的相关纯化操作，可以使用相同的缓冲系统。
4. 对于特殊的菌株，如果抽提效果不太理想，可以考虑进行冻融处理。冻融处理通常可进一步提高产品的蛋白抽提效果。
5. 如果使用本产品抽提得到的样品需要使用金属螯合纯化柱进行纯化，通常需要避免加入含EDTA 的试剂。但阿拉丁生产的His-tag Purification Resin(耐还原螯合型)可与 EDTA 兼容，因而在抽提试剂中含有EDTA仍然可用于含有His标签的蛋白纯化。
6. 本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。为了您的安全和健康，请穿实验服并戴一次性手套操作。
Bacterial Protein Extraction Reagent, also named Active Bacterial Protein Extraction Reagent, is a lysis buffer designed for isolation of native active bacterial proteins. It supports direct cell lysis and protein extraction with rapid processing speed, superior quality and high yield, eliminating the need for sonication or high-pressure homogenization. Optimized for endogenous soluble proteins of Escherichia coli and recombinant proteins expressed in E. coli, this reagent is perfectly suited for high-throughput rapid screening of protein expression.
This reagent works well with freshly harvested bacterial cells and frozen cell pellets, and delivers particularly excellent extraction efficiency for frozen samples. The required reagent volume is calculated based on the weight of cell pellets. EDTA-free protease inhibitor cocktails, salts and reducing agents can be directly supplemented into the reagent system. Efficient protein extraction can be accomplished without adding exogenous lytic enzymes.The reagent recovers soluble proteins and removes debris and contaminants attached to the surface of inclusion bodies to prepare high-purity inclusion body samples. Note that it cannot solubilize inclusion bodies; it only serves as a washing reagent to purify intact inclusion bodies.It is applicable to a broad range of biochemical and molecular biology applications, and compatible with routine downstream assays including protein purification, ELISA and Western blot.For protein purification: Ni-TED resins are recommended for His-tagged protein purification. If Ni-IDA, Ni-NTA, GST-tagged or MBP-tagged proteins are to be purified, the lysate is advised to be diluted 10-fold or desalted by dialysis before purification.
Protocols
1. Mini-scale Soluble Protein Extraction
Mini-scale extraction is used to verify target protein expression and expression level prior to preparative large-scale extraction.
1.1 Culture recombinant strains and induce target protein expression following standard culture protocols.
1.2 Take 1.5 mL bacterial suspension with OD₆₀₀ ranging from 0.5 to 2.0, centrifuge at 12,000–16,000 g for 2 min at 4 °C or ambient temperature, then discard supernatant.
1.3 Resuspend cell pellet in 0.2–0.4 mL bacterial protein extraction reagent; briefly vortex to achieve complete resuspension, followed by incubation at room temperature for no less than 1 min.
1.4 Centrifuge at 12,000–16,000 g (16,000 g preferred) for 5 min at 4 °C.
1.5 Carefully aspirate supernatant containing soluble protein. Avoid touching the pellet during supernatant collection to prevent contamination with excessive non-specific impurities.
1.6 Determine the localization of target protein (soluble supernatant vs insoluble pellet) via SDS-PAGE or Western blot analysis. Recommended loading volume per sample for SDS-PAGE: 5–15 μL.
2. Large-scale Soluble Protein Extraction
2.1 Culture recombinant strains and induce target protein expression per standard procedures.
2.2 Harvest 250 mL bacterial culture at OD₆₀₀ ≈ 2.0, centrifuge at 5,000 g for 10 min at 4 °C or ambient temperature; approximately 1 g wet cell pellet is obtained after supernatant removal. The pellet can be lysed immediately or frozen for later lysis. Freeze-thaw treatment generally improves total protein yield but may impair the bioactivity of certain sensitive proteins.
2.3 Resuspend pellet with 20–50 mL Bacterial Protein Extraction Reagent per gram of wet cells (supplement with appropriate protease inhibitors before use), and homogenize by repeated pipetting. Less reagent yields higher protein concentration with compromised extraction efficiency; excess reagent improves lysis efficiency while lowering final protein concentration. Minimal reagent volume is recommended when abundant wet cell pellets are available. For optimal results, supplement with nuclease or recombinant universal nuclease to a final concentration of 50 U/mL to reduce lysate viscosity.
2.4 Incubate at room temperature for no less than 1 minute; the incubation time can be extended appropriately to achieve complete protein extraction.
2.5 Centrifuge at 12,000–16,000 g (16,000 g preferred) for 10 min at 4 °C.
2.6 Gently collect supernatant with soluble protein; contact with cell pellet during aspiration introduces high levels of miscellaneous proteins.
2.7 Identify target protein distribution in supernatant or insoluble pellet by SDS-PAGE or Western blot; recommended sample loading volume: 5–15 μL per well for SDS-PAGE.
Troubleshooting
1. Low yield of target protein
(1) Incomplete bacterial lysis. Repeated freeze‑thaw cycles or supplementation with lysozyme facilitates cell rupture and improves protein recovery.
(2) Excessively high sample viscosity. Addition of nuclease or recombinant universal nuclease reduces viscosity and enhances soluble protein extraction.
(3) Target protein degradation. Supplement protease inhibitors to mitigate proteolysis.
(4) Poor intrinsic expression level of target protein. Increase IPTG dosage, prolong induction duration or optimize induction temperature; alternatively verify plasmid construction or switch to alternative expression strains.
(5) Target protein is insoluble. Inspect the post‑centrifugation pellet to confirm inclusion body formation.
(6) Insufficient volume of extraction reagent. Appropriately increase the dosage of lysis reagent.
2. Turbid soluble protein lysate
(1) Under-dosed extraction reagent. Supplement additional extraction buffer accordingly.
(2) Inadequate bacterial lysis. Supplement lysozyme during extraction, which commonly clarifies the final protein solution.
(3) Overlong frozen storage of purified protein lysate. It is recommended to complete downstream assays within 1–2 weeks after protein extraction.
(4) Insufficient centrifugal force or insufficient spinning duration for supernatant‑pellet separation. Centrifuge at 12,000–16,000 g (16,000 g recommended) for 10 min at 4 °C, or extend centrifugation time / elevate relative centrifugal force.
(5) Aggregation of expressed target protein. Add glycerol to a final concentration of 40–50% to inhibit protein aggregation and precipitation; alternatively isolate target protein via ammonium sulfate precipitation followed by resolubilization.
(6) Reduced protein solubility at low temperature. Warm the sample to ambient temperature to improve solubility and clarify turbidity; increasing reagent dosage is another feasible remedy.
3. Highly viscous soluble protein lysate
(1) Insufficient addition of extraction reagent. Increase the usage of extraction reagent properly.
(2) Supplement nuclease or recombinant universal nuclease to decrease sample viscosity.
Precautions
1. Protease inhibitor cocktail (Cat. No. P776695, P665818) or 100 mM PMSF (Cat. No. P301906) is recommended for addition to this product to inhibit protein degradation.
2. Recombinant universal nuclease (Cat. No. O775185, rp221814) or standard nuclease (Cat. No. B401536) is recommended to reduce the viscosity of protein lysates.
3. This product adopts a Tris-HCl buffer system, and the extracted protein samples are compatible with purification procedures using the same buffer system.
4. For difficult-to-lyse strains with unsatisfactory extraction performance, freeze-thaw treatment is recommended, which generally further improves protein extraction efficiency.
5. If samples extracted with this reagent will be purified via metal chelation chromatography, EDTA-containing reagents should generally be avoided. However, Aladdin His-tag Purification Resin (reduction-resistant chelating type) is compatible with EDTA, so lysates containing EDTA can still be used for purification of His-tagged proteins.
6. This product is for scientific research use by professionals only. It shall not be used for clinical diagnosis, medical treatment, food or pharmaceutical production, nor stored in residential areas. Wear a lab coat and disposable gloves during operation for personal safety and health.
规格
别名
细菌活性蛋白质提取试剂、细胞裂解试剂
英文别名
Bacterial Active Protein Extraction Reagent | Cell lysis reagent
规格或纯度
BioReagent, 细菌抽提用, 1×
稳定性与储存
Store at 2-8℃ long term (12 months).
英文名称
Bacterial Protein Extraction Reagent
储存条件
2-8°C储存
运输条件
低温运输
产品线
生物产品
名称和识别符
分子类型
生物试剂/缓冲液
技术文档
高级数据
产品属性
| pH | 7.5 |
|---|
质检证书（CoA,COO,BSE/TSE 和分析图谱)
C of A & Other Certificates(BSE/TSE, COO):
输入批号以搜索分析图谱:
通过匹配包装上的批号来查找并下载产品的 COA，每批产品都进行了严格的验证，您可放心使用！
| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0737761 |
分析证书 | L1507331 |
| ZJ26F0737760 |
分析证书 | L1507331 |
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