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View additional product information for Human beta Amyloid (1-40) Recombinant Protein - FAQs (03138)
12 product FAQs found
Recommendations for Beta Amyloid Peptide Reconstitution:
(1) To induce peptide aggregation for neurotoxicity studies: The appearance of toxicity has recently been shown to correlate to the extent of beta sheet structure (S. Wang et al. [2001] J. Biol. Chem. 276(45):42027-42034). Recommended preincubation is: Dissolve the lyophilized peptide in 0.1% (v/v) trifluoroacetic acid in water at 10 mg/mL. Dilute the peptide to 0.5-1.0 mg/mL with PBS (without Ca2+). Incubate at 25°C for 24-48 hrs (24-36 hrs is usually sufficient). Neurotoxic activity is usually observed at 30-100 micrograms/mL.
(2) To prepare peptide for studies which require minimal peptide aggregation: Dissolve the peptide at a concentration of 1 mg/mL in 100% HFIP (1,1,1,3,3,3-hexafluoro-2-propanol [Sigma-Aldrich Cat. No. 325244, 99.8% ACS reagent grade]). Incubate at RT for 2 hours. During the incubation, vortex the peptide solution several times at moderate speed, allowing the HFIP to cover as much of the surface area as possible. Dry down the HFIP/peptide solution under a gentle stream of nitrogen gas. Continue drying for an additional 10 minutes. Cap vial immediately. Resuspend the peptide in 100% DMSO. Incubate the peptide plus DMSO for 12 minutes at RT with periodic vortexing at moderate speed. Add 10 microliters of this DMSO/peptide solution dropwise to 10 mL of BSAT-DPBS (see formulation below) while vortexing at moderate speed. Peptides prepared in this manner have been used as standards in ELISA for the detection of beta amyloid in biological samples. When using this peptide in ELISA, it is important that the assay buffer used with the peptide standards has the same composition as the samples under investigation.
Buffer Formulations: DPBS Solution (10X Stock) (Gibco#14200-075) Dulbecco's PBS (DPBS w/o Mg2+, Ca2+) BSAT-DPBS Solution: 1X DPBS, pH 7.4 5% BSA 0.03% Tween-20
Note: The inclusion of a protease inhibitor (i.e. AEBSF [Sigma Cat. No. A-8456]) is recommended when the BSAT-BPBS solution is to be used for the analysis beta amyloid-containing biological samples. To prepare a 40 mM stock solution of AEBSF, add 100 mg of AEBSF to 10 mL DPBS, pH 7.4, containing 5% BSA. The stock solution should be diluted to a concentration of 1 mM AEBSF in BSAT-DPBS just prior to use.
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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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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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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.
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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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)).