Recombinant human GRP78 BiP protein (Active) (ab78432)

Reviews (1)Q&A (4)References (9)
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

Description

  • Product name

    Recombinant human GRP78 BiP protein (Active)
    See all GRP78 BiP proteins and peptides

  • Biological activity

    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.

  • Purity

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

  • Expression system

    Escherichia coli

  • Accession

    P11021

  • Protein length

    Full length protein

  • Animal free

    No

  • Nature

    Recombinant

    • Species

      Human

    • Sequence

      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

    • Predicted molecular weight

      78 kDa

    • Amino acids

      1 to 654

    • Tags

      His tag N-Terminus

    • Additional sequence information

      his-tagged (Gene ID 3309)(NM_005347)

Specifications

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.

  • Applications

    Western blot

    ELISA

    Functional Studies

    Competitive Binding Assays

    SDS-PAGE

  • Form

    Liquid
  • Concentration information loading...

Preparation and Storage

  • Stability and Storage

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

    Constituents: 0.32% Tris HCl, 0.37% Potassium chloride, 0.05% Magnesium chloride, 0.02% (R*,R*)-1,4-Dimercaptobutan-2,3-diol

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

General Info

  • Alternative names

    • 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

  • Function

    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.

  • Involvement in disease

    Autoantigen in rheumatoid arthritis.

  • Sequence similarities

    Belongs to the heat shock protein 70 family.

  • Cellular localization

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

Images

  • 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

Protocols

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

References (9)

Publishing research using ab78432? Please let us know so that we can cite the reference in this datasheet.

ab78432 has been referenced in 9 publications.

  • Rodríguez-Muñoz M  et al. The ALS-related s1R E102Q Mutant Eludes Ligand Control and Exhibits Anomalous Response to Calcium. Int J Mol Sci 21:N/A (2020). PubMed: 33020464
  • Singh P  et al. PSTi8 with metformin ameliorates perimenopause induced steatohepatitis associated ER stress by regulating SIRT-1/SREBP-1c axis. Heliyon 6:e05826 (2020). PubMed: 33426334
  • Castro NP  et al. Sulforaphane Suppresses the Growth of Triple-negative Breast Cancer Stem-like Cells In vitro and In vivo. Cancer Prev Res (Phila) 12:147-158 (2019). PubMed: 30679159
  • Gupta AP  et al. Pancreastatin inhibitor activates AMPK pathway via GRP78 and ameliorates dexamethasone induced fatty liver disease in C57BL/6 mice. Biomed Pharmacother 116:108959 (2019). PubMed: 31108350
  • Hossain Z  et al. Discovery of pancreastatin inhibitor PSTi8 for the treatment of insulin resistance and diabetes: studies in rodent models of diabetes mellitus. Sci Rep 8:8715 (2018). PubMed: 29880906
  • Qin EY  et al. Neural Precursor-Derived Pleiotrophin Mediates Subventricular Zone Invasion by Glioma. Cell 170:845-859.e19 (2017). PubMed: 28823557
  • Cerezo M  et al. Compounds Triggering ER Stress Exert Anti-Melanoma Effects and Overcome BRAF Inhibitor Resistance. Cancer Cell 29:805-819 (2016). PubMed: 27238082
  • Biswas N  et al. Discovery of a novel target for the dysglycemic chromogranin A fragment pancreastatin: interaction with the chaperone GRP78 to influence metabolism. PLoS One 9:e84132 (2014). Functional Studies . PubMed: 24465394
  • Lee SJ  et al. Statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, potentiate the anti-angiogenic effects of bevacizumab by suppressing angiopoietin2, BiP, and Hsp90a in human colorectal cancer. Br J Cancer 111:497-505 (2014). PubMed: 24945998

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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).

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Submitted Jan 08 2014

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