IP裂解液

产品名称：Cell Lysis Buffer for IP (Mammalian Whole Cell)

品牌：Engibody

货号：IF6601

简介：IP专用裂解液，用于免疫沉淀实验细胞和组织样本的裂解

Form

Liquid

Storage instruction

Store at +4°C.

Highlights

• Optimized for compatibility with immunoprecipitation and pull-down assays

• Compatible with protein assays, reporter assays and immunoassay procedures

• Ready-made formula is effective for extracting cytoplasmic, membrane and nuclear proteins

• Does not liberate genomic DNA which can cause high sample viscosity

Protocols

Procedure for Lysing Cell Monolayer (Adherent) Cultures

1. Carefully remove culture medium from cells. Wash the cells once with ice cold phosphate-buffered saline.

2. Add ice cold lysis buffer to the cells according to the table below and incubate on ice for 5-10 minutes with periodic mixing.

SizeVolume of Buffer

100 mm dish500-1,000 μL

60 mm dish250-500 μL

6-well plate200-400 μL per well

24-well plate100-200 μL per well

3. Transfer the lysate to a microcentrifuge tube and centrifuge at ~13,000 × g for 10 minutes to pellet the cell debris at 4°C.

4. Transfer supernatant to a new tube for protein concentration determination and further analysis.

Procedure for Lysing Cell Suspension Cultures

1. Centrifuge the cell suspension at 1,000 × g for 5 minutes to pellet the cells. Discard the supernatant.

2. Wash the cells once with ice cold PBS. Centrifuge at 1,000 × g for 5 minutes to pellet cells.

3. Add ice cold IP Lysis Buffer to the cell pellet. Use 500 μL of lysis buffer per 50 mg of wet cell pellet (10:1 v/w).

Note: If using a large amount of cells, first add 10% of the final volume of lysis buffer to the pellet and pipette the mixture up and down to mix. Add the remaining volume of lysis buffer to the cell suspension.

4. Incubate lysate on ice for 5-10 minutes with periodic mixing. Remove cell debris by centrifugation at ~13,000 × g for 10 minutes at 4°C.

5. Transfer supernatant to a new tube for protein concentration determination and further analysis.

Troubleshooting Guide

all reagents and lysates must be kept cold

Cell Lysis Buffer can be used for lysis of tissue samples, although a homogenization step is recommended after adding lysis buffer. Extract the tissue at a ratio of 100 mg of tissue to 1 ml of buffer. Sonication of the tissue lysate is also required.

The buffer does not contain protease or phosphatase inhibitors; Protease Inhibitor Cocktail or Phosphatase Inhibitor Cocktail should be added just before use to prevent proteolysis and maintain phosphorylation of proteins.

产品优势

•优化与免疫沉淀和下拉分析的兼容性

•与蛋白质分析、报告分析和免疫分析程序兼容

•现成配方可有效提取细胞质、膜和核蛋白

•不释放基因组DNA，这可能导致样品粘度过高

操作步骤

裂解细胞单层（粘附）培养物的步骤

1.小心地从细胞中取出培养基。用冰冷的磷酸盐缓冲盐水清洗细胞一次。

2.根据下表向细胞中加入冰冷的裂解缓冲液，并在冰上孵育5-10分钟，定期混合。

样本类型                  缓冲液体积

100 mm皿    500-1000μL

60 mm皿      250-500μL

6孔板           200-400μL/孔

24孔板         100-200μL/孔

3.将裂解液转移到微离心管中，并在约13000×g下离心10分钟，以在4°C下使细胞碎片沉淀。

4.将上清液转移到新试管中，用于蛋白质浓度测定和进一步分析。

裂解细胞悬浮培养物的步骤

1.以1000×g离心细胞悬浮液5分钟，使细胞沉淀。丢弃上清液。

2.用冰冷的PBS清洗细胞一次。以1000×g离心5分钟，使细胞沉淀。

3.向细胞颗粒中加入冰冷的IP裂解缓冲液。每50 mg湿细胞颗粒使用500μL裂解缓冲液（10:1 v/w）。

注：如果使用大量细胞，首先将最终体积的10%溶解缓冲液添加到颗粒中，并用移液管上下移动混合物以混合。向细胞悬浮液中加入剩余体积的裂解缓冲液。

4.在冰上孵育裂解物5-10分钟，并定期混合。在4°C下以约13000×g离心10分钟，去除细胞碎片。

5.将上清液转移到新试管中，用于蛋白质浓度测定和进一步分析。

常见问题解决指南

所有试剂和裂解物必须冷藏

细胞裂解缓冲液可用于组织样品的裂解，尽管建议在加入裂解缓冲液后进行均质化步骤。以100mg组织与1ml缓冲液的比例提取组织。还需要对组织裂解物进行超声处理。

缓冲液不含蛋白酶或磷酸酶抑制剂；蛋白酶抑制剂鸡尾酒或磷酸酶抑制剂鸡尾酒应在使用前添加，以防止蛋白质水解并维持蛋白质的磷酸化。