细胞核蛋白和胞浆蛋白提取试剂盒

  50t    ￥680

Nuclear and Cytoplasmic Extraction Kit

Product Number: NC-01    Size: 50T

Description

Nuclear and Cytoplasmic Extraction Reagents, sufficient reagents for extracting 50 cell pellet fractions having packed cell volumes of 50 µl each (a total of ~5.0 g of cell paste).

Storage: Upon receipt store kit components at 4°C. Product is shipped at ambient temperature. This product is guaranteed for one year from the date of purchase when handled and stored properly.

Introduction :

Nuclear and Cytoplasmic Extraction Reagents enable stepwise separation and preparation of cytoplasmic and nuclear extracts from mammalian cultured cells or tissue. Non-denatured, active proteins are purified in less than two hours. Addition of the first reagents to a cell pellet causes disruption of cell membranes and release of cytoplasmic contents.After recovering the intact nuclei from the cytoplasmic extract by centrifugation, the nuclei are lysed with the left reagents to yield the nuclear extract. Extracts obtained with this product generally have less than 10% contamination between nuclear and cytoplasmic fractions—sufficient purity for most experiments involving nuclear extracts.Use of nuclear  extracts is generally preferred to whole cell lysates for gene regulation studies. Cellular components present in whole cell lysates can adversely

affect nuclear protein interactions and stability, and nuclear proteins are more dilute in whole cell lysates than in specifically nuclear extracts. Nuclear extracts obtained with the Nuclear and Cytoplasmic Extraction Reagents are compatible with a variety of downstream applications including Western blotting,  the BCA Protein Assay，gel shift assays, reporter gene assays and enzyme activity assays.  

Important Procedural Notes   

• If using protease inhibitors cocktail (recommended), add them to reagents A and B .

• This procedure reflects reagent volumes based on samples with a packed cell volume of 50 µl (~40 mg of cell paste approximately equivalent to 2 x 106 HeLa cells). Determine the packed cell volume (or mass) of your samples, then substitute volumes as indicated in the following table.

• Perform all centrifugation steps at 4°C. Keep cell samples and extracts on ice.  

Procedure  

1. Isolate 50 µl packed cell volume (~100 mg) of cells in a 1.5 ml microcentrifuge tube by centrifugation at 500 x g for 2-3minutes.

    Note: Use one of the following methods to process a tissue sample:

• Cut the tissue into small pieces, add an appropriate buffer such as PBS, and homogenize in a tissue homogenizer. Pellet the cells by centrifugation at 500 x g for 2-3 minutes and remove the supernatant. Estimate the packed cell volume, add the appropriate amount of Reagent A according the chart on page 1, and continue with Step 4 below.

• Weigh the tissue, cut it into small pieces, dounce homogenize directly in Reagent A, and proceed to Step 4. Use a 10-fold excess of Reagent A over the weight of tissue (e.g. 500 µl Reagent A to 50 mg tissue). In Step 5 of the protocol, use 75 µl of Reagent B per 100 µl of Reagent A.

2. Using a pipet, carefully remove and discard the supernatant, leaving cell pellet as dry as possible.

3. Add 500 µl of ice-cold Reagent A to the cell pellet.

4. Vortex the tube vigorously on the highest setting for 15 seconds to fully resuspend the cell pellet. Incubate the tube on ice for 10 minutes.  

5. Centrifuge at 4˚C at 16,000 x g for 5 min.

6. Remove supernatant and keep it (this will contain everything except large plasma membrane pieces,DNA, nucleoli), extract out 10 µl for Bradford assay.

7. On ice resuspend pellet in 374 µl of Reagent B and add 26 µl of Reagent C (high salt helps lyse membranes and forces DNA into solution).

8 .Vortex on the highest setting for 15 seconds. Return the sample to ice and continue vortexing for 15 seconds every 10 minutes, for a total of 30 minutes.

9. Centrifuge the tube at maximum speed (~16,000 x g) in a microcentrifuge for 10 minutes.

10. Immediately transfer the supernatant (nuclear extract) fraction to a clean pre-chilled tube. Place on ice.

11. Store all extracts at -80°C until use.

Troubleshooting   

Origin:  Pioneer Biotechnology,Inc of China

Distributor: Pioneer Biotechnolgy,Inc

Contact:  Tel 029-88237772 or 13484545009  Fax 029-88237772

  E-mail : xfyangbio@163.com

          Internet: www.xfbio.com