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What methods of cell lysis are available? Product FAQ

Answer

Historically, physical lysis was the method of choice for cell disruption and extraction of cellular contents; however, it often requires expensive, cumbersome equipment and involves protocols that can be difficult to repeat due to variability in the apparatus (such as loose-fitting compared with tight-fitting homogenization pestles). Also, traditional physical disruption methods are not conducive for high-throughput and smaller volumes typical of modern laboratory research.
In recent years, detergent-based cell lysis methods have become the norm. Through empirical testing by trial and error, different detergent-based solutions composed of particular types and concentrations of detergents, buffers, salts and reducing agents have been developed to provide the best possible results for particular species and types of cells. Detergents have both lysing and solubilizing effects.

Answer Id:: E12788

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What types of detergents are available for cell lysis? Product FAQ

Answer

Detergents can be denaturing or non-denaturing with respect to protein structure. Denaturing detergents can be anionic such as sodium dodecyl sulfate (SDS) or cationic such as ethyl trimethyl ammonium bromide. These detergents totally disrupt membranes and denature proteins by breaking proteinprotein interaction. These detergents are considered harsh. Non-denaturing detergents can be divided into nonionic detergents (i.e., Triton X-100), bile salts (i.e., cholate), and zwitterionic detergents (i.e., CHAPS). These detergents do not denature proteins and do not break protein-protein interactions. These detergents are considered mild.

Answer Id:: E12790

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How does detergent-based cell lysis work? Product FAQ

Answer

Detergents are amphipathic molecules, meaning they contain both a nonpolar “tail” having aliphatic or aromatic character and a polar “head”. Like the components of biological membranes, detergents have hydrophobic-associating properties as a result of their nonpolar tail groups. Nevertheless, detergents are themselves water soluble.

Consequently, detergent molecules allow the dispersion (miscibility) of water-insoluble, hydrophobic compounds into aqueous media, including the extraction and solubilization of membrane proteins. Detergent monomers solubilize membrane proteins by partitioning into the membrane bilayer. With increasing amounts of detergents, membranes undergo various stages of solubilization.

Answer Id:: E12791

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What extraction reagents are recommended for efficient mouse tissue analysis? Product FAQ

Answer

We have 5 different cell and tissue extraction buffers suitable for preparing mouse cell and tissue extracts. These buffers can be used to extract cells and tissues from many other species as well. The exact compositions of all of our buffers are proprietary, but they are similar to those described by many researchers.

Four of these buffers can be used to prepare extracts which can be analyzed with our ELISA and Luminex kits and by Western blotting. Our Cell Extraction Buffer (FNN0011) contains extra phosphatase inhibitors and resembles the RIPA formulation that many people use. Our Tissue Extraction Reagents I (FNN0071) and II (FNN0081) contain different concentrations of NaCl and different surfactants, but are otherwise similar to each other. For those who prefer using an extraction buffer containing the detergent NP-40, we have our NP-40 Lysis Buffer (FNN0021). Finally, we sell a Denaturing Cell Extraction buffer (FNN0091) which contains 3 detergents and a chaotropic agent. Extracts prepared with FNN0091 can be analyzed with our ELISA kits and by Western blotting only. These buffers do not contain protease inhibitors, which the investigator should add right before use.

Answer Id:: E5224

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What are the components of a typical lysis buffer and why are they important? Product FAQ

Answer

Typical cell lysis reagents contain a detergent for protein solubilization and stabilization, a buffer to improve the extraction efficiency and stabilization of the protein, and a salt to improve solubility and stability and to facilitate selective compartment extraction.

Additional components can include reducing agents, chelators, crowding agents, and protease inhibitors.

Answer Id:: E12792

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Which lysis buffer should I choose for use for protein isolation using Dynabeads magnetic beads? Product FAQ

Answer

Detergent choice is important and may be influenced by the antigen's subcellular location and whether you would like to preserve subunit associations and other protein-protein interactions. The ionic strength (salt concentration), choice of detergent, and pH of the lysis buffer may affect the efficiency as well as the integrity of the target antigen.

Two commonly used buffers for cell lysis are:

Radioimmunoprecipitation assay (RIPA) buffer, useful for nuclear membrane disruption for nuclear extracts. It gives a lower background in immunoprecipitation but can denature some proteins. RIPA buffer is not recommended when studying protein-protein interactions, as it can disrupt these interactions.

NP-40 buffer, typically used for the study of protein-protein interactions, as it denatures proteins to a lesser extent. NP-40 is a nonionic detergent and is the most commonly used detergent in cell lysis buffers for immunoprecipitation and western blotting.

Answer Id:: E6002

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What can interfere with the EnzChek Myeloperoxidase (MPO) Activity assay? Product FAQ

Answer

MPO is inhibited by azide, diclofenac, methimazole, quercetin, rutin, and salicylhydroxamic acid. Make certain that samples do not include sodium azide. Endogenous catalases can interfere with the assay; catalase activity may be inhibited by using 3-amino-1,2,4-triazole. Detergents, common components in cell lysis buffers, should be avoided; use freeze/thawing and/or mechanical methods to lyse cells.

Answer Id:: E10809

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What is the advantage of using M-PER Mammalian Protein Extraction Reagent (Cat. No. 78501)? Product FAQ

Answer

The complete cell lysis reagent contains a mild, non-denaturing detergent in 25 mM Bicine buffer (pH 7.6), that dissolves cell membranes to extract and solubilize total protein from most cellular compartments. Extraction is accomplished in only 5 minutes and requires little or no additional mechanical disruption. M-PER Reagent is formulated for minimal interference with downstream biological applications.

Answer Id:: E13398

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Do you recommend a specific method for preparing samples for IP, Co-IP or pulldown assays? Product FAQ

Answer

Depending on your specific immunoprecipitation application and location of the target antigen within the cell (i.e., nucleus, cytosol, membrane, etc.,), we offer a variety of different lysis buffers, depending on the sample type and target (http://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-purification-isolation/cell-lysis-fractionation.html). You should develop an appropriate lysis strategy to maximize structural integrity. The ionic strength (salt concentration), choice of detergent, and pH of the lysis buffer may affect the immunoprecipitation efficiency as well as the integrity of the target antigen. Some common lysis buffers used include RIPA buffer and NP-40 buffer.

Answer Id:: E12994

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Development of improved cell lysis, solubilization and imaging approaches for proteomic analyses. Citations & References

  • Authors: Leimgruber RM, Malone JP, Radabaugh MR, LaPorte ML, Violand BN, Monahan JB
  • Journal: Proteomics
  • PubMed ID: 11840559
Catalog #

Why does the method of cell lysis matter? Product FAQ

Answer

Cell lysis is the first step in cell fractionation, organelle isolation, and protein extraction and purification. As such, cell lysis opens the door to a myriad of proteomics research methods. Many techniques have been developed and used to obtain the best possible yield and purity for different species of organisms, sample types (cells or tissue), and target molecule or subcellular structure. Subcellular fractionation and protein enrichment are important methods in the rapidly growing field of proteomics. Isolation of subcellular fractions and concentration of proteins in low abundance allow for more efficient identification and study of proteins of interest. Examples are the isolation of integral membrane proteins and nuclear proteins.

Answer Id:: E12789

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Is there a cell lysis procedure that will liberate microsome-bound proteins, which can then be purified on the ProBond column? Product FAQ

Answer

The addition of 0.2% Sarkosyl to the 6M guanidine lysis buffer should solubilize everything and may still be compatible with purification on ProBond columns. Generally, anionic detergents are incompatible with nickel-chelating columns, but up to 0.2% Sarkosyl has been successfully used in some cases.

Answer Id:: E3667

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Can you explain how the B-PER Bacterial Protein Extraction Reagent lyses cells? Product FAQ

Answer

The B-PER Reagent solution contains a proprietary, mild, non-ionic detergent in 20 mM Tris-HCl, pH 7.5. It effectively disrupts cells and solubilizes native or recombinant proteins without denaturation. The reagent creates holes in the cell membrane that will leak out cytosolic proteins. The sample may become very viscous when the bacterial chromosome is released. We recommend adding DNAse I (Cat. No. 90083) to the reagent to reduce viscosity. For better lysis efficiency and if there are inclusion bodies, we recommend adding Lysozyme (Cat. No. 90082) to the reagent. Alternatively, you may purchase the B-PER Bacterial Protein Extraction Reagent with Enzymes Kit (Cat. No. 90078 or 90079) that includes the B-PER Bacterial Protein Extraction Reagent, DNase I, and Lysozyme.

Answer Id:: E18353

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Do you have a protocol for preparing tissue homogenate and cell lysates to be used in the identification of connexins with immunoprecipitation and Western blot analysis? Product FAQ

Answer

The following is the tissue homogenate and cell lysate preparation protocol that we use in-house for various connexins:

(A) Tissue homogenate preparation for connexins 26, 29, 30, 32, 43, 47

-Remove tissues from the animal, snap freeze on dry ice and weigh. These can be stored at –80 degrees C until ready for use.
-Rapidly thaw tissues in 10 volumes ice-cold homogenization buffer (IP buffer) and homogenized in a glass-Teflon homogenizer (12 up and down strokes at ~600 RPM).
-Place homogenates on ice for about 15 min and then sonicate 2 x 10 s with a 10 s cooling period (on ice) between bursts.
-Determine the protein concentration using a detergent and Tris-compatible protein assay.
-Note: If DNA and membrane aggregates are a problem, simply spin down homogenate (10,000 x g, 3 min) and use the supernatant.

Homogenization buffer (IP buffer)
50 mM TRIS pH 8.0
10 mM MgCl2
150 mM NaCl
1% (v:v) Triton-X-100
Just before use add 1 mM sodium orthovanadate, 1 mM PMSF, and 2 mg/ml each of pepstatin, leupeptin and apoprotinin.

(B) Tissue homogenate preparation for Cx36 (retina)

-Dissect rat retina, snap-freeze and weigh as mentioned above.
-Homogenize retina as mentioned above but using 10 volumes of HEPES buffer pH 7.2 instead of the IP Buffer.
HEPES buffer
20 mM HEPES pH 7.2
100 mM NaCl
2 mM EDTA
Just before use add 0.5 mM PMSF and protease and phosphatase inhibitors as above (if necessary).

(C) Cell lysis

-Grow cells to desired confluence
-Rinse in ice cold PBS or HBSS and remove all the buffer
-Add 0.5-1.0 mL ice-cold IP Buffer (as mentioned above in A) and let sit for a few minutes on ice before scraping cells into a 1.5 mL (No Suggestions) tube
-Pass cells through a 26-28-gauge needle to sheer DNA (not required unless using the lysate for immunoprecipitation)
-Place the lysate on ice for about 30 min
-Centrifuge at 10,000 x g (tabletop centrifuge) for 5 min
-Collect supernatant and determine protein concentration using a detergent and Tris-compatible protein assay

Answer Id:: E4668

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Free fatty acids are produced in and secreted from target cells very early in cytotoxic T lymphocyte-mediated killing. Citations & References

  • Authors: Richieri GV, Kleinfeld AM
  • Journal: J Immunol
  • PubMed ID: 1918994
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