Dynabeads® M-500 Subcellular -"DISCONTINUED" - FAQs

Here is a sampling of references for organelle isolation, including vesicles and exosomes.

Dynabeads products used for exosome isolation via an antibody plus some other references where Dynabeads M-500 Subcellular has been used to isolate other types of organelles:

Rabesandratana H et al. (1998) Decay-accelerating factor (CD55) and membrane inhibitor of reactive lysis (CD59) are released within exosomes during in vitro maturation of reticulocytes. Blood 91:2573-2580.

Allan BB et al. (2001)Rab1 recruitment of p115 into a cis-SNARE complex: programming budding COPII vesicles for fusion. Science 289:444-448.

Li X et al. (2001) Na+-H+ exchanger 3 (NHE3) is present in lipid rafts in the rabbit ileal brush border: a role for rafts in trafficking and rapid stimulation of NHE3. J Physiol 537:537-552.

Some references for isolation of endosomes and lysosomes.

Gagescu R (2000) The recycling of endosome of Madin-Darby canine kidney cells is a mildly acidic compartment rich in raft components. Mol Biol Cell 11:2775-2791.

Provoda CJ et al. (2000) Evidence for a primary endocytic vesicle involved in synaptic vesicle biogenesis. J Biol Chem 275:7004-7012.

Tjelle TE et al. (1998) Degradation of phagosomal components in late endocytic organelles. J Cell Sci 111:141-148.

Vitale N et al. (1998) Localization of ADP-ribosylation factor domain protein (ARD1) in lysosomes and Golgi apparatus. Proc Natl Acad Sci USA 95:8613-8618.

Clayton A et al. (2001) Analysis of antigen presenting cell derived exosomes, based on immuno-magnetic isolation and flow cytometry. J Immunol Methods 247:163-174.

Marchand S et al. (2001) Differential targeting of components of the dystrophin complex to the postsynaptic membrane. Eur J Neurosci 13: 221-229.

Some references to articles where vesicles transporting from the ER have been isolated and related references:

Allan BB et al. (2000) Rab1 recruitment of p115 into a cis-SNARE complex: programming budding COPII vesicles for fusion. Science 289:444-448.

Seemann J et al. (2002) Partitioning of the matrix fraction of the Golgi apparatus during mitosis in animal cells. Science 295:848-851.

Jesch SA, Linstedt AD (19980 The Golgi and endoplasmic reticulum remain independent during mitosis and HeLa cells. Mol Biol Cell 9:623-635.

Sinai AP et al. (1997) Association of host cell endoplasmic reticulum and mitochondria with the Toxoplasma gondii parasitophorous vacuole membrane: a high affinity interaction. J Cell Sci 110:2117-2128.

Sans N et al. (2001) Synapse-associated protein 97 selectively associates with a subset of AMPA receptors early in their biosynthetic pathway. J Neurosci 21:7506-7516.

Abo-Hashema KAH et al. (1999) Evidence for triacylglycerol synthesis in the lumen of microsomes via a lipolysis-esterification pathway involving carnitine acyltransferases. J Biol Chem 274:35577-35582.

References where Dynabeads magnetic beads have been used to isolate mitochondria and other organelles, or to isolate mitochondrial or chloroplast DNA using Dynabeads magnetic beads:

Lüers GH et al. (1998) Immuno-isolation of highly purified peroxisomes using magnetic beads and continuous immunomagnetic sorting. Electrophoresis 19:1205-1210.

Sexton PS, Cenedella RJ. (2002) Immunomagnetic capture of lens membrane fractions containing steroid binding protein. Biochem Biophys Res Commun 295:1027-1031.

Hansen GH et al. (2001) Lipid rafts exist as stable cholesterol-independent microdomains in the brush border membrane of enterocytes. J Biol Chem 276:32338-32344.

Sinai AP et al. (1997) Association of host cell endoplasmic reticulum and mitochondria with the Toxoplasma gondii parasitophorous vacuole membrane: a high affinity interaction. J Cell Sci 110:2117-2128.

Jones SM et al. (1998) Immunoisolation of organelles using magnetic solid supports, p. 12-14 in Cell Biology: A Laboratory Handbook, 2nd edn., Academic Press.

Müsch A et al. (1997) Myosin II is involved in the production of constitutive transport vesicles from the TGN. J Cell Biol 138:291-306.

Herrnstadt C et al. (1999) A novel mitochondrial DNA-like sequence in the human nuclear genome. Genomics 60:67-77.

Kausch AP et al. (1999) Organelle isolation by magnetic immunoabsorption. BioTechniques 26 (2):336-343.

Hopwood AJ et al. (1996) DNA typing from human faeces. Int J Legal Med 108:237-243.

Marchington DR et al. (1997) Homopolymeric tract heteroplasmy in mtDNA from tissues and single oocytes: support for a genetic bottleneck. Am J Hum Genet 60:408-416.

Piercy R et al. (1993) The application of mitochondrial DNA typing to the study of white Caucasian genetic identification. Int J Legal Med 106:85-90.

Fangan BM et al. (1994) A general approach for PCR-amplification and sequencing of chloroplast DNA from crude vascular plant and algal tissue. BioTechniques 16:484-494.

Ferris C et al. (1995) Using chloroplast DNA to trace postglacial migration routes of oaks into Britain. Mol Ecol 4:731-738, 1995.

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Protein purity is most often determined by SDS-PAGE; sometimes, it is analyzed by HPLC as well. Please refer to either the product manual, data sheet or certificate of analysis (CoA) for product-specific purity levels and methods.

Cross-reactivity depends on the protein and species. In general, human proteins are often active on mouse cells. Many mouse proteins are active on human cells, but they may show a different specific activity than the human protein.

The ED50 value is defined as the protein concentration that shows a 50% maximal response.

ED50 values are provided as a range because of variability in the bioassay used to determine ED50 values. For calculations using ED50, we recommend using the midpoint of the range.

For additional information on ED50, please refer to our Tech Tip: https://assets.thermofisher.com/TFS-Assets/BID/Technical-Notes/converting-ed50-ng-ml-specific-activity-units-mg-tech-note.pdf

Yes, cell culture medium containing low concentrations of proteins can be filter-sterilized if needed. We recommend the use of a low-protein-binding 0.22 micron filter.

The specific activity of a bioactive protein can be determined using the following equation:
1 x 10E6 / ED50 (ng/mL) = specific activity (units/mg)

The ED50 can be found on the CoA for the recombinant protein, but we advise determining the ED50 of a given recombinant protein in your particular functional assay system.

For additional information on ED50, and its relationship with specific activity, please refer to our Tech Tip: https://assets.thermofisher.com/TFS-Assets/BID/Technical-Notes/converting-ed50-ng-ml-specific-activity-units-mg-tech-note.pdf

In general, we recommend storing lyophilized recombinant proteins at -20 degrees C upon arrival. However, short-term storage at 4 degrees C (up to 6 months) or at room temperature (up to 30 days) is permissible.

For reconstituted protein solutions, we recommend storing working aliquots (containing at least 10 µL of protein solution with carrier protein) at -20 degrees C to -80 degrees C for extended storage. Do not allow multipe freeze-thaw cycles.

As these storage conditions are protein-dependent, we do recommend checking the product-specific storage recommendations that are provided in either the product manual, data sheet, or certificate of analysis (CoA).

No, cell culture usage requires biologically active proteins. Recombinant proteins with known biological activity are indicated by the application “Functional Assay (FN)” on the product web page and data sheet. Whether a particular protein has been tested for bioactivity can be found on its product page. To find proteins with the FN designation, filter by Application on the recombinant proteins search page: https://www.thermofisher.com/antibody/protein/filter/type/Recombinant

Generally, a 0.1% solution of BSA is used when making dilute solutions of Gibco recombinant proteins and Gibco PeproTech recombinant proteins, but sometimes, as as high as 1% BSA is used. Protein solutions should be stored with carrier proteins (e.g., BSA) for extended storage, as it improves protein stability.

The same growth factors used for ES cell study can also be used for iPS study:

For cell maintenance: Human recombinant activin A, FGF-basic, and IGF-II(Stem Cells 24:1476 (2006); Nature 448:1015 (2007)).

For human ES cell differentiation: BMP-4, EGF, and HGF (Proc Natl Acad Sci U S A 97:11307 (2000)).

Information pertaining to whether a specific product has been tested against the WHO Reference Standard can typically be located on the product page or Certificate of Analysis (COA).

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For cell maintenance: Human recombinant activin A, FGF-basic, andIGF-II(Stem Cells 24:1476 (2006); Nature 448:1015 (2007)).

For human ES cell differentiation: BMP-4, EGF, and HGF(Proc Natl Acad Sci U S A 97:11307 (2000)).

Information pertaining to whether a specific product has been tested against the WHO Reference Standard can typically be located on the product page or Certificate of Analysis (COA).

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

There is no direct correlation or calculation between specific activity unit and International Unit (IU) values. International Units (IU) express a quantification of activity for the base amount of a substance in relation to an analogous reference standard with an internationally accepted unit of biological potency (i.e., IU/ng) that has been assigned based on an International Collaborative Study conducted by the World Health Organization (WHO). WHO Reference Standards are made available by the National Institute for Biological Standards and Control (NIBSC). Intended to simplify the comparison of activity of a substance obtained from different sources, IU measurements can vary as comparison methods are rarely the same between sources. A true direct comparison requires standardized methods of analysis in order to guarantee comparability of the substance’s activity in relation to its mass across sources.

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With a few exceptions, most human cytokines are active on mouse cells. Many mouse cytokines are active on human cells, but may show lower specific activity than the corresponding human cytokine. The interferons, GM-CSF, IL-3, and IL-4 are known to be species-specific with very little, if any, activity on non-homologous cells. In contrast, the FGFs and neurotrophins are very highly conserved and show excellent activity on cells of other animal species.

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Gibco recombinant proteins are frequently formulated without carrier proteins or additives (e.g., BSA, HSA, sucrose, etc.) and Gibco PeproTech recombinant proteins don't contain a carrier protein. As a result, during lyophilization, the protein product may be deposited on the vial as a thin, and sometimes invisible film instead of a pellet. The size of the pellet, if any, is not directly related to the quantity of the recombinant protein in the vial. Our quality control procedures assure that each vial contains the correct amount of product.

To ensure complete recovery of protein product, before opening a vial of lyophilized recombinant protein, we recommend centrifuging it in a microcentrifuge for 20-30 seconds to drive any protein that may be lodged in the cap or on the side or the bottom of the vial. After reconstitution, you can confirm the presence of product protein by running a small amount on SDS-PAGE. In general, a protein band with expected size should be visible with as little as 10 ng of protein loaded on an acrylamide gel.

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For PeproTech animal origin-free and PeproGMP proteins, the endotoxin level is guaranteed to be less than 0.01 ng/µg of protein, or 0.1 EU/µg. For most PeproTech RUO proteins, the endotoxin level is guaranteed to be less than 0.1 ng/µg of protein or 1 EU/µg. However, for many proteins, the actual measured endotoxin values are consistently below these stated endotoxin levels. Please contact our Quality Assurance department (peprotech.qualityassurance@thermofisher.com) for more information.

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Unless otherwise mentioned on the product information sheet, all PeproTech recombinant proteins are formulated in such a manner that the lyophilized proteins are very stable at room temperature. However, we recommend storing lyophilized products at -20 degrees C to -80 degrees C.

For reconstituted solutions of most recombinant proteins, we recommend short-term storage at 4 degrees C. For extended storage, the protein solution should be stored with a carrier protein (e.g., 0.1% BSA) in working aliquots and stored at -20 degrees C to -80 degrees C. Aliquots should be prepared to a concentration no lower than 1 µg/mL, and contain at least 10 µL volume, independent of concentration.

Please keep in mind that every freeze/thaw cycle may cause some denaturation of the protein; therefore, we do not recommend subjecting aliquots to more than a single freeze/thaw cycle.

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PeproTech recombinant proteins and PeproGMP cytokines are shipped at ambient temperature as they are lyophilized, helping to ensure that they are stable at a wide range of temperatures. Upon request and at an additional cost, these products can be shipped on ice packs or dry ice.

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Protein sequence information is available on the individual product page, except for proprietary engineered proteins.

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In general, we recommend using polypropylene tubes for storing aliquots of reconstituted recombinant proteins. Specific information for appropriate storage and handling of many recombinant proteins can be found on the product pages.

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No, each lot can vary slightly with regards to its specific activity. The information on the data sheet for each product is provided as a guide. For lot-specific information, please please check the Certificate of Analysis or contact Technical Support.

The ED50 for each functionally active recombinant protein can be found on its product page under Activity as well as in the data sheet. The specific activity (units/mg) for each recombinant protein can be determined from the ED50 using information available in our Tech Tip here: https://assets.thermofisher.com/TFS-Assets/BID/Technical-Notes/converting-ed50-ng-ml-specific-activity-units-mg-tech-note.pdf

The majority of our recombinant proteins have a guaranteed shelf life of one year unless otherwise indicated in our technical data sheets. This guarantee applies only if the recombinant protein is stored under the conditions stated in the data sheet. If you are not completely satisfied with the performance of the product, please contact Technical Support at techsupport@thermofisher.com for assistance.

If you carried out a test as described in our product insert and did not see any response, this could be due to several possibilities listed below:

- Protein was not reconstituted according to the instructions.
- The reconstituted protein is too old or protein might have precipitated. We recommend using the reconstituted protein within 3-6 months after reconstitution.
- Carrier protein was not added for proteins reconstituted at a concentration <0.1 mg/mL. Working solutions at <0.1 mg/mL should be used immediately; we do not recommend long-term storage of solutions at this concentration.
- The protein solution was exposed to multiple freeze/thaw cycles or was exposed to high temperature.
- Proteins were handled in the wrong types of vessels (some proteins are very sticky to certain plastics).

The solubility issue might be due to improper handling, or use of a solvent other than the one we recommended. We recommend that you warm the lyophilized powder to room temperature before you open the vial, and that you solubilize the protein in the buffer solution recommended in the manual (some proteins are more soluble in low pH buffer). Do not reconstitute at a protein concentration greater than 1 mg/mL. Do not vortex or mix protein solutions vigorously. Allowing the reconstituted protein to incubate overnight at 4 degrees C may help resolve any solubility issues.

Where possible, Thermo Fisher Scientific obtains International Unit (IU) values through multiple side-by-side comparisons of our product(s) against the analogous WHO Reference Standard within our biological activity assay. Performing multiple comparison tests allows us to account for any outliers due to possible variations with the assay (e.g., product handling, assay protocol, etc.). Using the results of these comparisons, we can provide a reliable quantification of our product’s activity in relation to the activity of the WHO Reference Standard.

Information pertaining to whether a specific product has been tested against the WHO Reference Standard can typically be located on the product page or COA.

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Bioassays are intended to measure the biological activity of a given growth factor or cytokine. In most cases, the bioassays are cell-based tests using different indicator cells such as primary cells or cell lines. The most commonly used bioassays include cell proliferation assay, chemotaxis assay, cytokine production assay, and cytotoxicity assay. The biological activity of a given cytokine is expressed as ED50, which represents the concentration of the cytokine that induces 50% of the maximum response. This method of expressing potency should only be used for cytokines whose dose-response curves are sigmoidal in shape.

In most cases, glycosylation of a growth factor or cytokine does not affect how it binds to a receptor directly, so its biological activity is not significantly affected by glycosylation in in vitro studies. However, the glycosylated protein is usually less sensitive to protease degradation and exhibits much longer half life in vivo than the same protein without glycosylation. Therefore, for in vivo studies, selecting a recombinant protein produced in a mammalian expression system or insect expression system might be a better choice than the same recombinant protein produced in E. coli.

The recombinant proteins provided by Thermo Fisher Scientific are usually produced in different expression systems such as E. coli, insect cells, or mammalian cells. The major differences in recombinant proteins produced in different expression systems are in the post-translational modifications present, such as glycosylation. Recombinant proteins produced in E. coli are not glycosylated. Recombinant proteins produced in insect cells are partially glycosylated without galactose and sialic acid and not branched. Recombinant proteins produced in mammalian cells are fully glycosylated.

Note: Mimic Sf9 Insect Cells (a derivative of the Sf9 insect cell line that has been modified to stably express a variety of mammalian glycosyltransferases) can be used for production of complex N-glycans with terminal sialic acid and galactose.

Repeated freeze/thaws will affect the stability of the recombinant protein. For example, freezing will significantly affect the pH of the protein solution and might cause denaturation of the protein (Arch Biochem Biophys 384:398 (2000)).

Depending on the product, the instructions for reconstituting the lyophilized protein are provided in either the product manual, product data sheet or Certificate of Analysis (COA), which can be found on the product page. We recommend that the container be first centrifuged to concentrate the powder at the bottom of the tube. Most proteins can be reconstituted with the addition of sterile, distilled water. However, the product data sheet or COA will indicate when a diluent other than water is required. Recommended solutions, carrier protein concentrations and extended storage conditions can also be found within these documents.

Reconstitution to a concentration of 0.1 to 1.0 mg/ml is recommended. For example, for 100 µg of protein, the amount of water that should be added should be between 100 µL and 1 mL, resulting in a protein solution with a concentration between 1 mg/mL and 0.1 mg/mL.

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Carrier proteins help improve the stability of proteins in dilute solutions, extending storage. Protein solutions are generally not very stable when frozen at low concentration. Upon freeze and thaw, some proteins in the solution may stick to the wall of the container, resulting in significant reduction of protein concentration if the starting concentration was low. Carrier proteins are used to reduce such loss. The most commonly used carrier proteins include bovine serum albumin (BSA), human serum albumin (HSA), or fetal bovine serum (FBS). These carrier proteins are generally used at 0.1% concentration. As a rule of thumb, if the concentration of the recombinant protein is less than 0.5 mg/mL, it is a good idea to add some carrier protein.