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查看更多产品信息 Human GDNF Recombinant Protein - FAQs (PHC7041, PHC7043, PHC7045, PHC7044)
43 个常见问题解答
我们的重组蛋白是以冻干粉的形式运送的。在大多数情况下管中可见蛋白粉末沉淀，有些时候粉末沉淀会脱离原位，粘附在管盖或管壁上。短暂的离心就可将蛋白粉末沉淀收集于管底。不过，并非所有的冻干蛋白均为肉眼可见。在大多数情况下，可见的白色粉末沉淀是原始缓冲溶液中的盐分，管中粉末沉淀大小与重组蛋白的量之间可能并无直接的相关性。如果重组蛋白在冻干前使用了不含盐分的溶剂，则可能并不容易看到粉末沉淀（通常为透明的）。您可通过在SDS PAGE上电泳少量的重溶蛋白溶液来确定蛋白的存在。一般来说，只需在丙烯酰胺胶中上样10ng蛋白，就可清晰地观察到确定预期大小的蛋白条带。
分析时间至关重要。每一种分析都需要进行条件优化并在响应的峰值时间进行。不同种类的细胞对于生长因子或细胞因子的响应可能会有差异。我们建议推荐用户按照产品手册中的推荐方法，使用相同的指示细胞重复我们的质控（QC）分析，来观察是否能够获得类似的反应结果。此外，血清也可能掩盖细胞的响应情况。对某些类型分析实验可能需要对细胞进行血清饥饿处理。
如果您按照我们产品插页中的描述进行了测试，但未见到任何反应，则可能由以下几种原因导致：
- 蛋白未按照说明书推荐方式进行溶解。
- 溶解后的蛋白放置时间过长或蛋白发生沉淀。我们推荐您在重新溶解后的3-6个月内使用这些蛋白产品。
- 当重新溶解的蛋白溶液浓度低于0.1 mg/mL时，未添加载体蛋白。低于0.1 mg/mL的工作液应立即使用；我们不推荐长期储存此浓度的蛋白溶液。
- 蛋白溶液经过多次冻融循环或暴露于高温条件下。
- 使用错误类型的容器操作蛋白（某些蛋白非常容易粘附于某些塑料器皿上）。
溶解性问题可能由操作不当，或未使用推荐溶剂所导致。我们推荐您在打开试剂管前将冻干粉平衡至室温，并使用产品手册中推荐的缓冲液溶解蛋白产品（某些蛋白在低pH缓冲体系中溶解得更好）。请勿配制使用蛋白浓度大于1 mg/mL的浓度溶解蛋白产品。请勿剧烈震荡或混合蛋白溶液。将重新溶解的蛋白置于4°C条件下孵育过夜可能有助于解决任何可溶性问题。
生物分析法常被用来检测某一特定生长因子或细胞因子的生物学活性。在大多数情况下，研究人员一般基于各类指示性细胞——如某些原代细胞或细胞系——来开展生物分析。最为常用的生物分析法包括细胞增殖分析(https://www.thermofisher.com/content/dam/LifeTech/Documents/PDFs/Cell-Proliferation-Assay.pdf)，细胞趋化性分析(https://www.thermofisher.com/content/dam/LifeTech/Documents/PDFs/Chemotaxis-Assay.pdf)，细胞因子生成分析(https://www.thermofisher.com/content/dam/LifeTech/Documents/PDFs/Cytokine-Production-Assay.pdf)和细胞毒性分析(https://www.thermofisher.com/content/dam/LifeTech/Documents/PDFs/Cytotoxicity-Assay.pdf)。通常以ED50来表示某一细胞因子的生物活性，该值代表了能够诱发50%最大效应的细胞因子浓度。这一表示效价的方法仅适用于那些剂量效应曲线为S形的细胞因子。
在大多数情况下，生长因子或细胞因子的糖基化作用不会直接影响该蛋白与受体之间的结合。所以在体外研究中，重组生长因子或细胞因子的生物活性不会受糖基化作用的显著影响。不过，糖基化蛋白通常对蛋白酶的降解更具耐受性，所以在体内的半衰期比无糖基化的同种蛋白更长。因此在体内研究中，选择哺乳动物表达系统或昆虫表达系统生产的重组蛋白可能比大肠杆菌中生产的同种重组蛋白效果更好。
Thermo Fisher Scientific所提供的重组蛋白产品通常在大肠杆菌，昆虫细胞或哺乳动物细胞等不同表达系统中产生。不同表达系统之间产生的重组蛋白的主要差别在于这些蛋白所携带的翻译后修饰，如糖基化修饰。在大肠杆菌中生成的重组蛋白未经糖基化。在昆虫细胞中产生的重组蛋白通常有部分糖基化，但不含半乳糖和唾液酸，糖链也无复杂分支。在哺乳动物细胞中产生的重组蛋白会带有完全的糖基化修饰。
备注：Mimic Sf9 昆虫细胞（来源于sf9昆虫细胞系，经改造可稳定表达多种哺乳动物糖基转移酶）可用于表达带末端唾液酸和半乳糖的复杂的N-糖链。
反复冻融会对重组蛋白的稳定性造成影响。举例来说，冰冻会显著影响蛋白溶液的pH值，可能会导致蛋白变性（（Arch Biochem Biophys 384:398 (2000)）。
溶解后的蛋白可按照工作用量进行分装。工作用量一般在10或20 μL左右。分装后的溶液应储存于聚丙烯管中。聚丙烯非常适合此种应用，因为蛋白不会强烈粘附于这种材质上。蛋白会粘附玻璃或聚苯乙烯材质，因此我们建议不要使用这些材质的储存容器来储存蛋白溶液。分装后的溶液可保存于–20°C条件下。如果您有–80°C冰箱可供使用，则这一温度条件更适于储存。
数据表中提供了重新溶解冻干蛋白的说明。我们推荐首先对盛装冻干蛋白的容器进行离心，以将这些粉末聚集在到管底。大多数蛋白可通过加入无菌蒸馏水来重新溶解。对于100 μg蛋白，所需加水量应在100 µL至1 mL之间，从而形成1 mg/mL至与0.1 mg/mL的蛋白溶液。如果需要除水以外的其他稀释剂，产品分析表中会对其进行说明。
蛋白溶液在低浓度的冰冻条件下冻存时通常不是很稳定。在冻融过程中，溶液中的某些蛋白会粘附在容器壁上，如果蛋白的起始浓度较低，则上述原因可能会导致造成蛋白浓度的进一步显著下降。因此，载体蛋白就是用于减少此类损失的。最为常用的载体蛋白包括牛血清白蛋白（BSA），人血清白蛋白（HSA）或胎牛血清（FBS）。这些载体蛋白通常的使用浓度是在0.1%。依根据经验数据，如果重组蛋白的浓度低于0.5 mg/mL，则推荐加入一些载体蛋白。
冻干蛋白通常可在2–8°C条件下储存数周，或在–20°C条件下长期保存。
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.
This product has not been tested to determine if it is cell culture grade.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
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).
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
Protein sequence information is available on the individual product page, except for proprietary engineered proteins.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
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.
Gibco recombinant proteins are frequently formulated without carrier proteins or additives (e.g., BSA, HSA, sucrose, etc.) and no Gibco PeproTech recombinant protein contains 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 to 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.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
Assay time is critical. Each assay needs to beoptimized and performed at the peak response time. Different cells may respond differently to a growth factor or cytokine. We suggest repeating our QC assay using same indicator cells as suggested in the manual to see if you can obtain a similar response. In addition, serum may be masking the response. Serum starvation might be needed for certain types of assays.
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.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
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)).
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, which results in significant reduction of protein concentration if the starting concentration was low. Therefore, 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
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.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
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.
Lyophilized proteins can typically be stored at 2 to 8 degrees C for several weeks, or stored dessicated at -20 degrees C for long-term storage.