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重组人GRP78 BiP蛋白(Active) (ab78432)

Reviews (1)Q&A (4)References (15)
SDS-PAGE - Recombinant human GRP78 BiP protein (Active) (ab78432)
  • SDS-PAGE - Recombinant human GRP78 BiP protein (His tag) (ab78432)

Key features and details

  • Expression system: Escherichia coli
  • Purity: > 90% SDS-PAGE
  • Active: Yes
  • Tags: His tag N-Terminus
  • Suitable for: WB, ELISA, Functional Studies, Competitive Binding Assays, SDS-PAGE

描述

  • 产品名称

    重组人GRP78 BiP蛋白(Active)
    参阅全部 GRP78 BiP 蛋白酶

  • 生物活性

    ab78432 has ATPase activity at the time of manufacture of 2.3µM phosphate liberated/hr/µg protein in a 200µl reaction at 37°C (pH7.5) in the presence of 20ul of 1mM ATP, using a Malachite Green assay.

  • 纯度

    > 90 % SDS-PAGE.
    Ab78432 is purified by affinity purification.

  • 表达系统

    Escherichia coli

  • Accession

    P11021

  • 蛋白长度

    Full length protein

  • 无动物成分

    No

  • 性质

    Recombinant

    • 种属

      Human

    • 序列

      MKLSLVAAML LLLSAARAEE EDKKEDVGTV VGIDLGTTYS CVGVFKNGRV EIIANDQGNR ITPSYVAFTP EGERLIGDAA KNQLTSNPEN TVFDAKRLIG RTWNDPSVQQ DIKFLPFKVV EKKTKPYIQV DIGGGQTKTF APEEISAMVL TKMKETAEAY LGKKVTHAVV TVPAYFNDAQ RQATKDAGTI AGLNVMRIIN EPTAAAIAYG LDKREGEKNI LVFDLGGGTF DVSLLTIDNG VFEVVATNGD THLGGEDFDQ RVMEHFIKLY KKKTGKDVRK DNRAVQKLRR EVEKAKRALS SQHQARIEIE SFYEGEDFSE TLTRAKFEEL NMDLFRSTMK PVQKVLEDSD LKKSDIDEIV LVGGSTRIPK IQQLVKEFFN GKEPSRGINP DEAVAYGAAV QAGVLSGDQD TGDLVLLDVC PLTLGIETVG GVMTKLIPRN TVVPTKKSQI FSTASDNQPT VTIKVYEGER PLTKDNHLLG TFDLTGIPPA PRGVPQIEVT FEIDVNGILR VTAEDKGTGN KNKITITNDQ NRLTPEEIER MVNDAEKFAE EDKKLKERID TRNELESYAY SLKNQIGDKE KLGGKLSSED KETMEKAVEE KIEWLESHQD ADIEDFKAKK KELEEIVQPI ISKLYGSAGP PPTGEEDTAE KDEL

    • 预测分子量

      78 kDa

    • 氨基酸

      1 to 654

    • 标签

      His tag N-Terminus

    • 额外的序列信息

      his-tagged (Gene ID 3309)(NM_005347)

技术指标

Our Abpromise guarantee covers the use of ab78432 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

  • 应用

    Western blot

    ELISA

    Functional Studies

    Competitive Binding Assays

    SDS-PAGE

  • 形式

    Liquid
  • Concentration information loading...

制备和贮存

  • 稳定性和存储

    Shipped at 4°C. Store at -20°C. Stable for 12 months at -20°C.

    pH: 7.50
    Constituents: 0.79% Tris HCl, 0.88% Sodium chloride, 10% Glycerol (glycerin, glycerine)

    This product is an active protein and may elicit a biological response in vivo, handle with caution.

常规信息

  • 别名

    • 78 kDa glucose regulated protein
    • 78 kDa glucose-regulated protein
    • AL022860
    • AU019543
    • BIP
    • D2Wsu141e
    • D2Wsu17e
    • Endoplasmic reticulum lumenal Ca(2+)-binding protein grp78
    • Endoplasmic reticulum lumenal Ca2+ binding protein grp78
    • Epididymis secretory sperm binding protein Li 89n
    • FLJ26106
    • Glucose Regulated Protein 78
    • Glucose Regulated Protein 78kDa
    • GRP 78
    • GRP-78
    • GRP78
    • GRP78_HUMAN
    • Heat shock 70 kDa protein 5
    • Heat Shock 70kDa Protein 5
    • Heat shock protein family A (Hsp70) member 5
    • HEL S 89n
    • Hsce70
    • HSPA 5
    • HSPA5
    • Immunoglobulin Heavy Chain Binding Protein
    • Immunoglobulin heavy chain-binding protein
    • mBiP
    • MIF2
    • Sez7
    see all

  • 功能

    Probably plays a role in facilitating the assembly of multimeric protein complexes inside the endoplasmic reticulum. Involved in the correct folding of proteins and degradation of misfolded proteins via its interaction with DNAJC10, probably to facilitate the release of DNAJC10 from its substrate.

  • 疾病相关

    Autoantigen in rheumatoid arthritis.

  • 序列相似性

    Belongs to the heat shock protein 70 family.

  • 细胞定位

    Endoplasmic reticulum lumen. Melanosome. Cytoplasm. Identified by mass spectrometry in melanosome fractions from stage I to stage IV.
  • Information by UniProt

图片

  • SDS-PAGE - Recombinant human GRP78 BiP protein (Active) (ab78432)
    SDS-PAGE - Recombinant human GRP78 BiP protein (Active) (ab78432)
    SDS-PAGE of 78kDa Grp78 (Bip) protein.
  • SDS-PAGE - Recombinant human GRP78 BiP protein (His tag) (ab78432)
    SDS-PAGE - Recombinant human GRP78 BiP protein (His tag) (ab78432)

    Lane 1. Molecular weight ladder

    lane 2. GRP78 BiP protein

实验方案

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

文献 (15)

发表研究结果有使用 ab78432？请让我们知道，以便我们可以引用本数据表中的参考文章。

ab78432 被引用在 15 文献中.

  • Zheng N  et al. M2 macrophage-derived exosomes suppress tumor intrinsic immunogenicity to confer immunotherapy resistance. Oncoimmunology 12:2210959 (2023). PubMed: 37197441
  • Allu PKR  et al. Functional Gly297Ser Variant of the Physiological Dysglycemic Peptide Pancreastatin Is a Novel Risk Factor for Cardiometabolic Disorders. Diabetes 71:538-553 (2022). PubMed: 34862200
  • Wei J  et al. Cucurbitacin B-induced G2/M cell cycle arrest of conjunctival melanoma cells mediated by GRP78-FOXM1-KIF20A pathway. Acta Pharm Sin B 12:3861-3876 (2022). PubMed: 36213538
  • Garzón-Niño J  et al. αN-Acetyl β-Endorphin Is an Endogenous Ligand of σ1Rs That Regulates Mu-Opioid Receptor Signaling by Exchanging G Proteins for σ2Rs in σ1R Oligomers. Int J Mol Sci 24:N/A (2022). PubMed: 36614024
  • Shin J  et al. Possible Involvement of Adipose Tissue in Patients With Older Age, Obesity, and Diabetes With SARS-CoV-2 Infection (COVID-19) via GRP78 (BIP/HSPA5): Significance of Hyperinsulinemia Management in COVID-19. Diabetes 70:2745-2755 (2021). PubMed: 34615619
View all Publications for this product

客户评价及客户问答

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1-5 of 5 Abreviews or Q&A

Circulating GRP78 Quantification using Sandwich ELISA

 Poor
Abreviews
abreview image
Application
ELISA
The following protocol was used to optimize a standard curve in an ELISA assay intended to quantify GRP78 human protein in blood serum samples:

Reagents:
Coating buffer for primary Antobody: Bicarbonate/carbonate coating buffer (100 mM) :
3.03 g Na2CO3,
6.0 g NaHCO3
1000 ml distilled water
Adjust to pH 9.6 (using NaOH or HCL)
 Washing buffer (aka. PBST): 0.05% Tween 20 in PBS (250µLTween 20, 655204, CALBIOCHEM) in 500mL PBS)
 Sample dilution buffer for GRP78 standard (ab78432, Abcam): PBS
 Blocking buffer: 5% Nonfat milk (dissolved in PBS)
 Capture Ab: Rat Ab (ab25192, Abcam), 0.5mg/mL, 200µL of 0.5 mg/mL rat anti-GRP78 mAbis diluted in 10mL PBS, where the final concentration is 10µg/mL (1:50 dilution)
 Detection Ab: Rabbit polyclonal against GRP78 (ab21685): 1:1,000 dilution. For each 96-well plate (High Binding 96 well plate [Immunogen, Thermo, S0611810]), 10 µL of Rabbit anti-GRP78 pAb (ab21685, Abcam) dissolved in 10mL PBS (final dilution is 1:1,000)
 Secondary Ab: Donkey anti rabbit-HRP, sc-2313, Santa Cruz, in PBS (1:8,000 dilution). For each 96-well plate, (1:8,000) 1.25 µL of Donkey anti-rabbit secondary Ab (sc-2313, Santa Cruz) dissolved in 10mL PBS
 Patient’s serum dilution: 1:50 dilution (20 µL sample is diluted in 980 µL PBS)
 GRP78 standard: GRP78 (ab78432, Abcam,50µg at 1000µg/mL): serial dilutions starting at 4000 pg/mL (4000, 2000, 1000, 500, 50, 10, 1 pg/mL)
 TMB solution
 TMB stopping solution (1N H2SO4).

Procedure

1. Add 100 µL of Rat anti-GRP78, 10µg/mL Mab (76-E6) (ab25192, Abcam) into each well in a high binding 96-well plate (Corner Notch, Thermo, 15041).
2. After adding 100 µL anti-GRP78 Mab, seal the plates and incubate overnight at 4°C.
3. Aspirate the anti-GRP 78 Mab antibodies out of the wells.
4. Wash each well with 200 µL of PBST (PBS + 0.05% Tween20) at 5 min x 3 times to remove unbounded anti-GRP78.
5. Add 200 µL 0.1% BSA (0.05g BSA in 50 mL PBS) to each well and incubate for 2 hrs at room temperature, or overnight at 4oC.
6. Remove the BSA and wash each well with 200 µL of PBST (PBS + 0.05% Tween20) at 5 min x 3 times to remove unbounded anti-GRP78.
7. Add samples (100 µL) that have been diluted 1:50 in PBS and GRP78 standard solution (100 µL) into each well in duplicates.
8. Incubate plate at 37 °C for 1.5 hrs on a shake and bake oven.
9. Remove samples via aspiration after incubation and wash each well with 200 µL PBST at 5 min X 3 times
10. Add 100 µL of detecting Ab (rabbit polyclonal against GRP78, ab21685, abcam, 1:1,000), seal plate and incubate for 2 hours 37.
11. Remove the rabbit polyclonal anti-GRP78 (ab21685) via aspiration and wash plate for 5 min X 4 times with PBST.
12. Add 100 µL of Secondary Ab (donkey anti rabbit-IgG-HRP, sc-2313, Santa Cruz, 1:8,000) into each well in the plate.
13. Seal plate and incubate at room temperature for 2hrs.
14. Discard Secondary Ab and wash plate for 5 min x 4 times with PBST.
15. Add the chromagen (100 µL of TMB (substrate) to the plate and incubate for15 min at 37 degrees Celsius (if blue color is too faint, increase incubation time).
16. Stop the reaction by adding 50 µL of 1N H2SO4 into each well and mix gently.
17. Check the absorbance at 450nm with Spectrophotometer.

Results: absorbance was the same across all concentrations of GRP78 protein standard (including background).
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

提交于 Jan 08 2014

Question

What grade/purity of ATP was used in the kit that was used to calulate the biological activity of the protein.

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Answer

Thank you for contacting Abcam.

The purity of ATP used in the kit used to calculate the biological activity of the protein was >99%.

I believe my colleagues have already emailed you with the catalogue number of the Malachite Green assay that was used during the testing.

If there is anything else I can help you with, please let me know.

Read More

Question

With that said, any further details of the assay conditions used to test the recombinant GRP78 activity would be very helpful. I am planning to purchase this item and probably the kit as well for the purpose of conducting activity assays, and your working protocol seems like a great place to start for my own experiments.

Thanks,

That actually helps a lot, to know that this particular assay kit (ab65622) was used to assess the ATPase activity of the recombinant GRP78 product (ab78432). Perhaps that information could be added to the product information for the GRP78?

thanks,

Read More
Answer

Thank you for your reply.

I apologize, however there was a misunderstanding. I did not know that you were asking for the malachite green based assay used for assessing the activity of ab78432.

We do not knowwith which kitthe recombinant GRP78ab78432 activity has been tested. We have now asked the laboratory to receive these details and will update you on this.

The ab65622 is the only malachite green based kit we have in our catalog currently - this is why I cited this product.

Once again, please apologize for the confusion. We will write you again, as soon as we have more information. Thank you for your patience.

Read More

Question

Customer contacted us thet are concerned that the presence of 20mM Sodium phosphate in the buffer will interfere with any activity assay. This product  has ATPase activity at the time of manufacture of 2.3µM phosphate liberated/hr/µg protein in a 200µl reaction at 37°C (pH7.5) in the presence of 20ul of 1mM ATP using a Malachite Green assay, was this prior to adding this buffer? They are concerned that as this is such a small volume how could they remove the sodium phosphate?

Read More
Answer

Thank you for contacting us.

I have found out the the buffer listed on the datasheet is incorrect. The buffer supplied does not have any sodium phosphate included and is instead:



20mM Tris,50mM KCl,5mM MgCl2,1mM DTT, pH7.5.



I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Question

How long is the His tag on this product?

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Answer

Thank you for contacting us. The His tags for all four proteins you asked about are all 6X tags. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.  

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