UltraBio™ Mammalian Active Protein Extraction Reagent

Item Number

M751612

• Product Name: UltraBio™ Mammalian Active Protein Extraction Reagent

Product Description

UltraBio™ Mammalian Active Protein Extraction Reagent, also known as UltraBio™ Mammalian Native Protein Extraction Reagent, allows for rapid extraction of cytoplasmic proteins, nuclear proteins and membrane proteins from mammalian cells or tissues. The extracted proteins are of high quality, high yield, and high activity.This product is a buffer (pH 7.6) containing a specific mild non-denaturing detergent to ensure that proteins can be extracted efficiently with activities well preserved, which facilitates subsequent assays. Its extraction effect is similar to Aladdin's Lysis Buffer for Western and IP or RIPA Lysis Buffer .Because the detergent contained in this product is relatively mild, its extraction efficiency for some insoluble proteins is relatively low. To obtain a relatively high yield of poorly soluble proteins, we recommend using extraction reagents containing strong detergents. The detergent in protein extracts obtained with this product can be removed by dialysis or ultrafiltration.This product can quickly, gently and effectively extract the total protein of mammalian cells or tissues. The extracted proteins can maintain their original spatial structures and biological activities, and can be used in a variety of biochemistry and cell biology applications such as western blot, immunoprecipitation, co-immunoprecipitation, ELISA (enzyme linked immunosorbent assay), reporter gene assay of luciferase, β-gal, alkaline phosphatase, and chloramphenicol acetyltransferase (CAT), activity assay of protein kinases such as PKA, PKC, and tyrosine kinase, and separation and purification of proteins with HA, Flag, Myc, V5, His, and GST tags.The protein concentration of sample lysate obtained from this product can be determined using Aladdin's BCA Protein Assay Kit or Bradford Protein Assay Kit (detergent compatible) .

Precautions：

All steps of protein extraction should be performed on ice or 4℃ to keep protein stable.Appropriate protease inhibitors and phosphatase inhibitors can be added to protein extraction reagent as needed. provides a variety of inhibitor cocktails, such as Protease and Phosphatase Inhibitor Cocktail for General Use , Protease and Phosphatase Inhibitor Cocktail for General Use, MS-safe , and Protease and Phosphatase Inhibitor Cocktail for Mammals . If phosphorylated proteins are not to be detected, it is unnecessary to use phosphatase inhibitors.ZonaseTM Super Nuclease (, D7121/D7126) can be supplemented in this product for protein extraction to reduce the viscosity of the crude extract.If the extracted His-tag recombinant protein is going to be purified with nickel column, EDTA should not be used generally for protein extraction, as EDTA will destroy most metal chelation purification resins including the Ni-NTA resin. However, His-tag Purification Resin that is EDTA compatible can be used to purifiy His-tagged proteins from protein extracts containing EDTA.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.

Instructions for Use：

1. Reagent preparation.Take an appropriate amount of Mammalian Active Protein Extraction Reagent, add appropriate amounts of protease and phosphatase inhibitors ( Super Nuclease ( Mammalian Active Protein Extraction Reagent by referring to Table 1, and incubate on ice for 2-10 minutes. Cells will be lysed immediately in the extraction reagent. For routine assays, proceed to the next step immediately. However, to obtain more proteins or for some proteins that are difficult to extract, continue to incubate on ice for 2-20 minutes (if necessary, shake to further improve the extraction effect) to allow the cells to be fully lysed, which can increase protein yields by 10-50%, depending on cells.Table 1. The amount of Mammalian Active Protein Extraction Reagent recommended for different culture vessels.Multiple Well Plates or DishesGrowth area (cm2)*Volume of Extraction Reagent (μl)10-cm dish55500-10006-cm dish21200-4006-well plate9.5100-20012-well plate3.850-10024-well plate1.925-5048-well plate0.9512.5-2596-well plate0.325-10c. Suspend the cell extract by pipetting and transfer it to a centrifuge tube. Centrifuge at 14,000×g at 4℃ for 5-10 minutes.d. Transfer the supernatant containing soluble proteins to a new centrifuge tube. Perform subsequent assays immediately or store the supernatant at -20℃ or -80℃ for future use. If necessary, keep the precipitate for subsequent analysis. Do not touch the pellet when aspirating the supernatant. 3. Protein extraction from suspension cells.a. Cell collection: Collect cells by centrifugation at 2500×g at 4℃ or room temperature for 10 minutes, and discard the supernatant. b. Cell washing (optional): Wash cells with an appropriate amount of PBS, centrifuge at 800×g for 5 min at room temperature, and discard the supernatant.. Note: If the culture medium and the added drugs have no impact on subsequent experiments, cell washing is not necessary. Otherwise, wash cells twice..c. Cell lysis: Aspirate the cell culture medium or wash buffer, add an appropriate amount of prechilled Mammalian Active Protein Extraction Reagent by referring to Table 1, and incubate on ice for 2-10 minutes. Cells will be lysed immediately in the extraction reagent. For routine assays, proceed to the next step immediately. However, to obtain more proteins or for some proteins that are difficult to extract, continue to incubate on ice for 2-20 minutes (if necessary, shake to further improve the extraction effect) to allow the cells to be fully lysed, which can increase protein yields by 10-50%, depending on cells.d. Suspend the cell extract by pipetting and centrifuge at 14,000×g at 4℃ for 5-10 minutes.e. Transfer the supernatant containing soluble proteins to a new centrifuge tube. Perform subsequent assays immediately or store the supernatant at -20℃ or -80℃ for future use. If necessary, keep the precipitate for subsequent analysis. Do not touch the pellet when aspirating the supernatant.4. Protein extraction from tissues.a. Tissue processing: Cut the tissue into small pieces, or grind it into powder after freezing with liquid nitrogen.b. Homogenization and extraction: Add 200-400μl of prechilled mammalian active protein extraction reagent per 20mg of tissue, homogenize thoroughly with a glass homogenizer or TissueMasterTM Handheld Homogenizer .c. Incubate on ice for 2-10 minutes. For routine assays, proceed to the next step immediately. However, to obtain more proteins or for some proteins that are difficult to extract, continue to incubate on ice for 2-20 minutes (if necessary, shake to further improve the extraction effect) to allow for sufficient lysis.d. Centrifuge at 14,000×g for 5-10min at 4℃.e. Transfer the supernatant containing soluble proteins to a new centrifuge tube. Perform subsequent assays immediately or store the supernatant at -20℃ or -80℃ for future use. If necessary, keep the precipitate for subsequent analysis. Do not touch the pellet when aspirating the supernatant.FAQ: 1. Protein yield is low.a. Insufficient cell or tissue lysis. Extend the incubation time on ice, shake appropriately during the incubation, or appropriately increase the amount of protein extraction reagents for protein extraction.b. Proteins were degraded during the extraction. Adding protease inhibitors in protein extraction reagent can inhibit protein degradation.c. Tissue homogenization is insufficient. Properly extend the homogenization time.d. The viscosity of the protein extract is too high. Increase the amount of protein extraction reagent or add Super Nuclease ( Super Nuclease, ≥99%100KUD7121-2000KU Super Nuclease, ≥99 00KUD7121-25KU Super Nuclease, ≥99%25KUD7121-500KU Super Nuclease, ≥99%500KUD7121-5KU Super Nuclease, ≥99%5KUD7126-100KU Super Nuclease, ≥99%, with His-tag100KUD7126-2000KU Super Nuclease, ≥99%, with His-tag2000KUD7126-25KU Super Nuclease, ≥99%, with His-tag25KUD7126-500KU Super Nuclease, ≥99%, with His-tag500KUD7126-5KU Super Nuclease, ≥99%, with His-tag5KUP0006CDetergent Compatible Bradford Protein Assay Kit800P0009Enhanced BCA Protein Assay Kit5000TP0010Enhanced BCA Protein Assay Kit500TP0010SEnhanced BCA Protein Assay Kit200TP0011BCA Protein Assay Kit5000TP0012BCA Protein Assay Kit500TP0012SBCA Protein Assay Kit200TP0013Cell Lysis Buffer for Western or IP100mlP0013BRIPA Lysis Buffer 100mlP0013CRIPA Lysis Buffer (Medium)100mlP0013DRIPA Lysis Buffer (Weak)100mlP0013ERIPA Lysis Buffer Set 50ml eachP0013GSDS Lysis Buffer100mlP0013JCell lysis buffer for Western and IP without inhibitors100mlP0013KRIPA lysis buffer 100mlP0013M-100ml Mammalian Active Protein Extraction Reagent100mlP0013Q-100ml Bacterial Active Protein Extraction Reagent100mlP0013Q-500ml Bacterial Active Protein Extraction Reagent500mlP0013S-100ml Insect Active Protein Extraction Reagent100mlP0027Nuclear and Cytoplasmic Protein Extraction Kit50TP0028Nuclear and Cytoplasmic Protein Extraction Kit100TP0033Membrane and Cytosol Protein Extraction Kit100TP1005Protease inhibitor cocktail for general use, 100X1mlP1006Protease inhibitor cocktail for general use, 100X5mlP1008Protease inhibitor cocktail for general use, MS-SAFE, 50X1mlP1009Protease inhibitor cocktail for general use, MS-SAFE, 50X5mlP1010Protease inhibitor cocktail for mammalian cell and tissue extracts, 100X1mlP1011Protease inhibitor cocktail for mammalian cell and tissue extracts, 100X5mlP1015Protease inhibitor cocktail for plant cell and tissue extracts, 100X1mlP1016Protease inhibitor cocktail for plant cell and tissue extracts, 100X5mlP1020Protease inhibitor cocktail for fungal and yeast extracts, 100X1mlP1021Protease inhibitor cocktail for fungal and yeast extracts, 100X5mlP1025Protease inhibitor cocktail for bacterial cell extracts, 100X1mlP1026Protease inhibitor cocktail for bacterial cell extracts, 100X5mlP1030Protease inhibitor cocktail for purification of His-tagged proteins, 100X1mlP1031Protease inhibitor cocktail for purification of His-tagged proteins, 100X5mlP1045Protease and phosphatase inhibitor cocktail for general use , 50X2mlP1046Protease and phosphatase inhibitor cocktail for general use, 50X10mlP1048Protease and phosphatase inhibitor cocktail for general use, MS-safe, 50X1mlP1049Protease and phosphatase inhibitor cocktail for general use, MS-safe, 50X5mlP1050Protease and phosphatase inhibitor cocktail for mammalian cell and tissue extracts, 50X2mlP1051Protease and phosphatase inhibitor cocktail for mammalian cell and tissue extracts, 50X10mlP1055Protease and phosphatase inhibitor cocktail for plant cell and tissue extracts, 50X2mlP1056Protease and phosphatase inhibitor cocktail for plant cell and tissue extracts, 50X10mlP1060Protease and phosphatase inhibitor cocktail for fungal and yeast extracts, 50X2mlP1061Protease and phosphatase inhibitor cocktail for fungal and yeast extracts, 50X10mlP1065Protease and phosphatase inhibitor cocktail for bacterial cell extracts, 50X2mlP1066Protease and phosphatase inhibitor cocktail for bacterial cell extracts, 50X10mlP2210 His-tag Purification Resin (耐还原螯合型)10mlP2210FTHis-tag Purification Resin2mlP2220 His-tag Purification Resin1000mlP2226His-tag Protein Purification Kit10mlS3090Cell and Tissue Lysis Buffer for Nitric Oxide Assay100mlST206Lysozyme (＞20KU/mg)0.5gST506PMSF(100mM)10ml