Recombinant Human HSP70/HSPA1A Protein, CF Summary
HSP70/HSPA1A is a molecular chaperone that assists in the folding of nascent polypeptides and the refolding of denatured proteins, but can also promote their degradation in conjunction with specific E3 ligases, such as CHIP, if either of these processes proceeds inefficiently. Reaction conditions will need to be optimized for each specific application. We recommend an initial HSP70/HSPA1A concentration of 2-3 μM for in vitro use. IMPORTANT: HSP40/DNAJB1 (Catalog # AP-110), or another suitable co-chaperone, is required for HSP70/HSPA1A activity and should be used at a concentration that is equimolar to HSP70/HSPA1A.
Product Datasheets
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins.Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration.The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard.In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
AP-100
| Formulation | Xmg/ml (X μM) in 50 mM HEPES pH 8, 100 mM NaCl, 5 mM DTT |
| Shipping | The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
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Reconstitution Calculator
Background: HSP70/HSPA1A
Heat Shock 70kDa Protein (HSP70), also known as Heat Shock 70kDa Protein 1A (HSPA1A), is a 641 amino acid (aa) member of the HSP70 family of molecular chaperones with a predicted molecular weight of 70 kDa. Human HSP70/HSPA1A shares 95% and 97% aa sequence identity with the mouse and rat orthologs, respectively. It has an N-terminal nucleotide-binding domain, which contains ATPase activity, and a C-terminal substrate-binding domain (1). HSP70/HSPA1A promotes the proper folding of nascent polypeptides and assists in the refolding of denatured proteins (2). However, if either of these processes proceeds too slowly or fails, HSP70/HSPA1A can interact with the HSP40 co-chaperone protein and the CHIP/STUB1 Ubiquitin ligase (E3) to promote ubiquitination and degradation of the nascent polypeptide or denatured protein (3,4). HSP70/HSPA1A can be regulated post-translationally via multiple mechanisms, including phosphorylation, ubiquitination, and methylation (5-8). For example, unmethylated HSP70/HSPA1A localizes to the cytoplasm, but following methylation on Lys561 it is found only in the nucleus (8). Pathologically, HSP70/HSPA1A has been implicated in the promotion of multiple cancer types (8-10). Conversely, it is thought to protect against several neurodegenerative diseases that are caused by the accumulation of misfolded proteins (11,12).
- Qi, R. et al. (2013) Nat. Struct. Mol. Biol. [Epub ahead of print].
- Kampinga, H.H. & E.A. Craig (2010) Nat. Rev. Mol. Cell Biol. 11:579.
- McDonough, H. & C. Patterson (2003) Cell Stress Chaperones 8:303.
- Donnelly, B.F. et al. (2013) J. Biol. Chem. 288:13124.
- Muller, P. et al. (2012) Oncogene [Epub ahead of print].
- Liu, M. et al. (2008) J. Biol. Chem. 283:35783.
- Moore, D.J. et al. (2008) J. Neurochem. 105:1806.
- Cho, H.S. et al. (2012) Nat. Commun. 3:1072.
- Wu, F.H. et al. (2012) Cancer Lett. 317:157.
- Gaudio, E. et al. (2013) Blood 121:351.
- Miyata, Y. et al. (2011) Future Med. Chem. 3:1523.
- Wang, A.M. et al. (2013) Nat. Chem. Biol. 9:112.
Citations for Recombinant Human HSP70/HSPA1A Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products.The data collected includes not only links to publications in PubMed,but also provides information about sample types, species, and experimental conditions.
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- March1-dependent modulation of donor MHC II on CD103+ dendritic cells mitigates alloimmunityAuthors: TJ Borges, N Murakami, FD Machado, A Murshid, BJ Lang, RL Lopes, LM Bellan, M Uehara, KH Antunes, MJ Pérez-Saéz, G Birrane, P Vianna, JIB Gonçalves, RF Zanin, J Azzi, R Abdi, S Ishido, JS Shin, APD Souza, SK Calderwood, LV Riella, C BonorinoNat Commun, 2018;9(1):3482.Species: MouseSample Types: Whole CellsApplications: Bioassay
- Toll-like receptor 4 signaling in trigeminal ganglion neurons contributes tongue-referred pain associated with tooth pulp inflammation.Authors: Ohara, Kinuyo, Shimizu, Kohei, Matsuura, Shingo, Ogiso, Bunnai, Omagari, Daisuke, Asano, Masatake, Tsuboi, Yoshiyuk, Shinoda, Masamich, Iwata, KoichiJ Neuroinflammation, 2013;10(0):139.Species: RatSample Types: Whole TissueApplications: IHC
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试剂盒里有详细的说明书，告诉你样品需要多少量，每个试剂需要加入多少量，和详细的实验步骤，一般买来就可以用，不用人教。
所以你问一个样需要多少量是没法回答的，测定过程是要加很多种试剂的。
1.模板提取（一般为RNA）：Trizol、氯仿、异丙醇、无水乙醇、DEPC处理水
2.模板浓度测定：分光光度计或NanoDrop
3.逆转录：逆转录试剂盒（或者一步法试剂盒），这一步可以用普通PCR做，也可以用水域做。
4.荧光定量PCR试剂：通常有用SYBR Green Mix做的，但是这里建议你用EvaGreen做，灵敏度和平行性都要好于SYBR Green，并且如果你那是ABI或者Stratagene的PCR如果用SYBR Green还需要加一步Rox很麻烦。
5.其他：除了以上的那些还需要离心管、PCR管或板（Axygen反应比较好）、移液枪等，暂时就想到这么多。
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只要引物设计的可以在基因组上做，那么理论上是没问题的！
只需要用RT-PCR荧光定量试剂盒里面的第二个，专门用来做Real-timePCR的那些试剂就可以了！
