產(chǎn)品名稱：Cell Lysis Buffer for CoIP (Mammalian Whole Cell)

品牌：Engibody

貨號(hào)：IF6603

簡介：CoIP專用裂解液，用于免疫共沉淀CoIP裂解樣本細(xì)胞

CoIP Lysis Buffer is used to lyse mammalian whole cells under nondenaturing conditions.

Gentle formulation helps maintain protein complexes for co-immunoprecipitation

Description

Cell Lysis Buffer for CoIP (Mammalian Whole Cell)

Tested applications

CoIP

Storage instruction

Store at +4°C.

Highlights

• Gentle formulation helps maintain protein-protein complexes for co-immunoprecipitation

• 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

CoIP裂解緩沖液用于在非變性條件下裂解哺乳動(dòng)物全細(xì)胞。

溫和的配方有助于維持蛋白質(zhì)復(fù)合物以進(jìn)行共免疫沉淀。

產(chǎn)品描述

CoIP的細(xì)胞裂解緩沖液（哺乳動(dòng)物全細(xì)胞）

經(jīng)過測試的應(yīng)用

CoIP免疫共沉淀

存儲(chǔ)說明

儲(chǔ)存溫度為+4°C。

產(chǎn)品優(yōu)點(diǎn)

•溫和的配方有助于維持蛋白質(zhì)-蛋白質(zhì)復(fù)合物，以進(jìn)行共免疫沉淀

•優(yōu)化與免疫沉淀和pulldown實(shí)驗(yàn)的兼容性

•與蛋白質(zhì)分析、報(bào)告分析和免疫分析實(shí)驗(yàn)兼容

•現(xiàn)成配方可有效提取細(xì)胞質(zhì)、膜和核蛋白

•不釋放基因組DNA，不會(huì)導(dǎo)致樣品粘度過高

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.

Size                Volume 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 CoIP 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

1. all reagents and lysates must be kept cold

2. 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.

3. 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.

Protocol

裂解細(xì)胞單層（粘附）培養(yǎng)物的程序

1.小心地從細(xì)胞中取出培養(yǎng)基。用冰冷的磷酸鹽緩沖鹽水清洗細(xì)胞一次。

2.根據(jù)下表向細(xì)胞中加入冰冷的裂解緩沖液，并在冰上孵育5-10分鐘，定期混合。

細(xì)胞類型              所需用量

100 mm培養(yǎng)皿   500-1000μL

60 mm培養(yǎng)皿   250-500μL

6孔板    200-400μL/孔

24孔板   100-200μL/孔

3.將裂解液轉(zhuǎn)移到微離心管中，并在約13000×g下離心10分鐘，以在4°C下使細(xì)胞碎片沉淀。

4.將上清液轉(zhuǎn)移到新試管中，用于蛋白質(zhì)濃度測定和進(jìn)一步分析。

裂解細(xì)胞懸浮培養(yǎng)物的步驟

1.以1000×g離心細(xì)胞懸浮液5分鐘，使細(xì)胞沉淀。丟棄上清液。

2.用冰冷的PBS清洗細(xì)胞一次。以1000×g離心5分鐘，使細(xì)胞沉淀。

3.將冰冷的CoIP裂解緩沖液添加到細(xì)胞顆粒中。每50 mg濕細(xì)胞顆粒使用500μl裂解緩沖液（10:1 v/w）。

注：如果使用大量細(xì)胞，首先將最終體積的10%溶解緩沖液添加到顆粒中，并用移液管上下移動(dòng)混合物以混合。向細(xì)胞懸浮液中加入剩余體積的裂解緩沖液。

4.在冰上孵育裂解物5-10分鐘，并定期混合。在4°C下以約13000×g離心10分鐘，去除細(xì)胞碎片。

5.將上清液轉(zhuǎn)移到新試管中，用于蛋白質(zhì)濃度測定和進(jìn)一步分析。

常見問題解答指南

1.所有試劑和裂解物必須冷藏

2.細(xì)胞裂解緩沖液可用于組織樣品的裂解，盡管建議在加入裂解緩沖液后進(jìn)行均化步驟。以100mg組織與1ml緩沖液的比例提取組織。還需要對組織裂解物進(jìn)行超聲處理。

3.緩沖液不含蛋白酶或磷酸酶抑制劑；蛋白酶抑制劑雞尾酒或磷酸酶抑制劑雞尾酒應(yīng)在使用前添加，以防止蛋白質(zhì)水解并維持蛋白質(zhì)的磷酸化。

僅用于科研，不作用人體