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technique(s)
protein extraction: suitable
application(s)
sample preparation
General description
Total protein extraction kit is an excellent tool for the initial purification and preparation of proteins from any tissues and cells. Total protein isolated by this kit is native and can be used for downstream applications including SDS-PAGE, Western blotting, gel mobility shift, protein assays and other procedures. The kit is easy to use: no scraping, no freeze-thaw cycles, and no sonication.
This kit contains enough reagents for the isolation of protein from 5 grams of tissue or cultured cells.
This kit contains enough reagents for the isolation of protein from 5 grams of tissue or cultured cells.
Application
Isolate protein from large samples <br /><br />
Isolate protein either from tissues or from cultured cells <br /><br />· Protein is intact, and some proteins purified by this kit could be over 200 kd in mass <br /><br />The isolated protein can be used for Western Blot, DNA-protein interaction, enzymatic activity analysis, protein-protein interaction, and immunoprecipitation and tissue specific expression identification. <br /><br />PROTOCOL: <br /><br />Important: <br /><br />Aliquot 50X PI on ice and store at -20°C. <br /><br />Always make fresh working solutions before the isolation of proteins. <br /><br />1. Dilute 50X PI solution to 1X PI in TM buffer keeping the solution on ice. <br /><br />2. Weigh certain tissues and chop them into small pieces. <br /><br />3. Keep the tissues on dry ice. <br /><br />4. Add 1X PI to the tissue at 2.5 mL per gram of tissue or per 25 million cells, and put in ice for 5 minutes. <br /><br />5. Homogenize the tissue or cells for 20 seconds and then put on dry ice for 15 seconds. <br /><br />6. Homogenize the tissue or cells for the second 20 seconds. (A third 20 seconds homogenization may be required if the tissue or cells are not well homogenized.) <br /><br />7. Rotate the homogenized tissue or cells at 4°C for 20 minutes. <br /><br />8. Centrifuge at 11,000 rpm at 4°C for 20 minutes. <br /><br />9. Collect the supernatant. <br /><br />10. Determine the concentration of the total protein.
Isolate protein either from tissues or from cultured cells <br /><br />· Protein is intact, and some proteins purified by this kit could be over 200 kd in mass <br /><br />The isolated protein can be used for Western Blot, DNA-protein interaction, enzymatic activity analysis, protein-protein interaction, and immunoprecipitation and tissue specific expression identification. <br /><br />PROTOCOL: <br /><br />Important: <br /><br />Aliquot 50X PI on ice and store at -20°C. <br /><br />Always make fresh working solutions before the isolation of proteins. <br /><br />1. Dilute 50X PI solution to 1X PI in TM buffer keeping the solution on ice. <br /><br />2. Weigh certain tissues and chop them into small pieces. <br /><br />3. Keep the tissues on dry ice. <br /><br />4. Add 1X PI to the tissue at 2.5 mL per gram of tissue or per 25 million cells, and put in ice for 5 minutes. <br /><br />5. Homogenize the tissue or cells for 20 seconds and then put on dry ice for 15 seconds. <br /><br />6. Homogenize the tissue or cells for the second 20 seconds. (A third 20 seconds homogenization may be required if the tissue or cells are not well homogenized.) <br /><br />7. Rotate the homogenized tissue or cells at 4°C for 20 minutes. <br /><br />8. Centrifuge at 11,000 rpm at 4°C for 20 minutes. <br /><br />9. Collect the supernatant. <br /><br />10. Determine the concentration of the total protein.
Components
TM Buffer 13 mL (Stored at 2-8°C) 50X PI Buffer 260 μL (Stored at -20°C)
Storage and Stability
Store the TM Buffer at 2-8°C for up to 12 months after date of receipt. Store the 50X PI Buffer at -20°C in undiluted aliquots for up to 12 months after date of receipt. Avoid repeated freeze/thaw cycles.
Disclaimer
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
signalword
Danger
Hazard Classifications
Aquatic Chronic 3 - Eye Dam. 1 - Met. Corr. 1 - Skin Irrit. 2
Storage Class
8A - Combustible, corrosive hazardous materials
wgk_germany
WGK 3
Certificates of Analysis (COA)
Search for Certificates of Analysis (COA) by entering the products Lot/Batch Number. Lot and Batch Numbers can be found on a product’s label following the words ‘Lot’ or ‘Batch’.
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