INSTRUCTIONS
六级翻译预测RIPA Buffer
89900 RIPA Buffer, 100mL
89901 RIPA Buffer, 250mL
Contents: 25mM Tris•HCl pH 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS
Storage: Upon receipt store at 4°C. Product shipped at ambient temperature.
Introduction
The Thermo Scientific RIPA buffer is one of the most reliable buffers ud to ly cultured mammalian cells from both plated cells and cells pelleted from suspension cultures. This buffer enab
寻梦园les protein extraction from cytoplasmic, membrane and nuclear proteins and is compatible with many applications, including reporter assays, protein assays, immunoassays and protein purification.
Important Product Information
•RIPA Buffer does not contain protea or phosphata inhibitors. If desired, add protea inhibitors, such as Thermo Scientific Halt Protea Inhibitor Cocktail (Product No. 78410) and Halt™ Phosphata Inhibitor Cocktail (Product No.
78420) to the reagent to prevent proteolysis and maintain phosphorylation status of proteins. Add protea and
phosphata inhibitors immediately before u.
•U 1mL of cold RIPA Buffer for every 5 × 106 of HeLa or A431 cells (~20µL of packed cells, which is equivalent to ~40mg of cells). To obtain concentrated protein extracts, directly ly cells on plate and u less buffer.
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•Some protein kinas and other enzymes may be nsitive to the components of the RIPA Buffer, resulting in their decread activity. In such cas, prepare a RIPA buffer that does not contain sodiu
m deoxycholate and SDS.
•RIPA Buffer is compatible with the Thermo Scientific Pierce BCA Protein Assay Kit (Product No 23225).
Procedure for Lysis of Monolayer-cultured Mammalian Cells
Note: If desired, add protea and phosphata inhibitors to the RIPA Buffer immediately before u.
1.Carefully remove (decant) culture medium from adherent cells.
2.Wash cells twice with cold PBS.
3.Add cold RIPA Buffer to the cells. U 1mL of buffer per 75cm2 flask containing 5 × 106 HeLa or A431 cells. Keep on
ice for 5 minutes, swirling the plate occasionally for uniform spreading.
4.Gather the lysate to one side using a cell scraper, collect the lysate and transfer to a microcentrifuge tube. Centrifuge
samples at ~14,000 ×g for 15 minutes to pellet the cell debris.
Note: To increa yields, sonicate the pellet for 30 conds with 50% pul.
5.Transfer supernatant to a new tube for further analysis.气管炎症状
Procedure for Lysis of Suspension-cultured Mammalian Cells
Note: If desired, add protea and phosphata inhibitors to the RIPA Buffer immediately before u.
1.Pellet the cells by centrifugation at 2500 ×g for 5 minutes. Discard the supernatant.
2.Wash cells twice in cold PBS. Pellet cells by centrifugation at 2500 ×g for 5 minutes.
3.Add RIPA Buffer to the cell pellet. U 1mL of RIPA buffer for 40mg (~5 × 106 of HeLa cells) of wet cell pellet. Pipette
the mixture up and down to suspend the pellet.
Note: To increa yields, sonicate the pellet for 30 conds with 50% pul.
4.Shake mixture gently for 15 minutes on ice. Centrifuge mixture at ~14,000 ×g for 15 minutes to pellet the cell debris.
5.Transfer supernatant to a new tube for further analysis.
带自的成语Troubleshooting
Problem Possible Cau Solution
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Low total protein yield Some cells are more resistant
to lysis than others Make sure the cell pellet is thoroughly suspended in RIPA Buffer and incubate for longer with occasional swirling − sonicate the pellet to increa yield
Low concentration of proteins Excess buffer ud U less buffer (e.g., 0.25-0.5mL per 75cm2 flask containing
5 × 10
6 cells) − u a sufficient amount to cover the entire plate
Proteolysis No protea inhibitors added Add Halt Protea Inhibitor Cocktail to the buffer before u
Low phosphorylation of proteins Phosphata activity Add Halt Phosphata Inhibitor Cocktail to the buffer before u Protein is non-phosphorylated
or poorly phosphorylated
None
Related Thermo Scientific Products
78410 Halt Protea Inhibitor Cocktail Kit
78420 Halt Phosphata Inhibitor Cocktail, 1mL
78248 B-PER® Bacterial Protein Extraction Reagent, 500mL
78990 Y-PER® Yeast Protein Extraction Reagent, 500mL
89826 Mem-PER® Membrane Protein Extraction Reagent Kit
78833 NE-PER® Nuclear and Cytoplasmic Extraction Kit
23227 Pierce® BCA Protein Assay Kit
26148 Pierce Direct IP Kit
34080 SuperSignal® West Pico Chemiluminescent Substrate, 500mL
34076 SuperSignal® West Dura Extended Duration Substrate, 200mL
General References
Cao, F., et al. (2005). Identification of an esntial molecular contact point on the duck hepatitis B virus rever transcripta. J Virol79(16):10164-70. Pfrepper, K.I. and Flugel R.M. (2005). Molecular characterization of proteolytic processing of the gap proteins of human spumaretrovirus. Methods in Mol Biol304:435-44.
Sefton, B.M. (2005). Labeling cultured cells with 32Pi and preparing cell lysates for immunoprecipitation. Unit 18.2. F. M. Ausubel, R. Brent, R.E.
感动作文450字
Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl (eds.) Current Protocols in Molecular Biology. John Wiley & Sons, Inc.
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