Anti-BiP (GRP78) Antibody, arginylated (Nt-Glu19) (C15-1317-793)

General description

78 kDa glucose-regulated protein (UniProt P11021; also known as BiP, Endoplasmic reticulum lumenal Ca(2+)-binding protein grp78, GRP-78, Heat shock 70 kDa protein 5, Immunoglobulin heavy chain-binding protein) is encoded by the HSPA5 (also known as GRP78) gene (Gene ID 3309) in human. BiP (GRP78 or heat shock 70 kDa protein 5), calreticulin (CRT), and protein disulphide isomerase (PDI) are ER lumen folding factors that, together with other PTM enzymes, assist the process of newly synthesized proteins before their release from ER. BiP, CRT, and PDI themselves are subject to posttranslational signal peptide removal after their synthesis, exposing E19, E18, and D18 at the newly formed N-terminus, respectively. The N-terminus D and E residues of the mature proteins are permissive to arginine-tRNA-protein transferase 1/ATE1-catalyzed arginylation, forming arginylated mature proteins (R-BiP, R-CRT, R-PDI) with altered cellular localization to allow their participation in non-ER functions. Although only the basal levels of R-CRT and R-PDI, but not R-BiP, are generally detectible in non-stimulated cells, upregulated N-terminal arginylation of all three proteins is observed upon cytosolic dsDNA exposure and proteasomal inhibition, indicating a shared role in innate immune responses to invading microbes. In addition, ER stress induction by thapsigargin treatment synergistically boosts proteasomal inhibition-induced upregulation of cellular levels of R-BiP, R-CRT, and R-PDI, suggesting that ER stress accelerates the supply of ER lumenal BiP, CRT, and DPI for N-terminal arginylation.

Specificity

This rabbit polyclonal antibody specifically detected the immunogen peptide, but not the control peptide without arginylation at the N-terminal Glu19 (Cha-Molstad, H., et al. (2015). Nat. Cell Biol. 17(7):917-929).

Immunogen

Epitope: N-terminus

Linear peptide corresponding to the N-terminal sequence of arginylated mature human BiP (GRP78).

Application

Detect arginylated mature BiP (GRP78) using this rabbit polyclonal Anti-BiP (GRP78), arginylated (Nt-Glu19), Cat. No. ABS2103, validated for use in Dot Blot, ELISA, Immunocytochemistry, and Western Blotting.

Research Category
Signaling

Western Blotting Analysis: 0.5 µg/mL from a representative lot detected Arg-BiP(19-651), but not Val-BiP(19-651) GFP fusion in 7.5 µg of lysate from MEF cells transfected to express either Ub-Arg-BiP(19-124) or Ub-Val-BiP(19-124) GFP fusion contruct co-translationally cleaved into Ub and Arg-BiP(19-124)-GFP or Val-BiP(19-124)-GFP by intracellular Ub hydrolases.

Immunocytochemistry Analysis: 10 µg/mL from a representative lot detected BiP Nt-Glu19 arginylation induction in (18-hr 3 µM MG132 and 200 nM thapsigargin) treated HeLa cells (Courtesy of Yong Tae Kwon, Ph.D. , Seoul National University, Korea).

Immunocytochemistry Analysis: 10 µg/mL from a representative lot detected BiP Nt-Glu19 arginylation induction in (18-hr 3 µM MG132 and 200 nM thapsigargin) treated wild-type, but not arginine-tRNA-protein transferase 1/ATE1-deficient, MEFs (Courtesy of Yong Tae Kwon, Ph.D. , Seoul National University, Korea).

Western Blotting Analysis: 0.2 µg/mL from a representative lot detected BiP Nt-Glu19 arginylation induction in (18-hr 3 µM MG132 and 200 nM thapsigargin) treated HeLa cells (Courtesy of Yong Tae Kwon, Ph.D. , Seoul National University, Korea).

Western Blotting Analysis: 0.2 µg/mL from a representative lot detected a target R-BiP(19-651)-GFP fusion band in MEF cells transfected to express Ub-R-BiP(19-651)-GFP or Ub-BiP(19-651)-GFP, but not Ub-V-BiP(19-651)-GFP. In ATE1-deficient MEFs, the target R-BiP(19-651)-GFP band was detected only when the cells were tranfected to express Ub-R-BiP(19-651)-GFP, but not Ub-BiP(19-651)-GFP (Courtesy of Yong Tae Kwon, Ph.D. , Seoul National University, Korea).

Dot Blot Analysis: A representative lot detected the immunogen peptide, but not the control peptide without arginylation at the N-terminal Glu19 (Cha-Molstad, H., et al. (2015). Nat. Cell Biol. 17(7):917-929).

ELISA Analysis: A representative lot detected the immunogen peptide, but not the control peptide without arginylation at the N-terminal Glu19 (Cha-Molstad, H., et al. (2015). Nat. Cell Biol. 17(7):917-929).

Immunocytochemistry Analysis: A representative lot detected poly(dA:dT) transfection-induced formation of BiP arginylation/R-BiP-positive puncta co-localized with those containing p62, LC3, and ubiquitin conjugates, while R-BiP and ER stainings are mutually exclusive (Cha-Molstad, H., et al. (2015). Nat. Cell Biol. 17(7):917-929).

Western Blotting Analysis: A representative lot detected the production of R-BiP(19-651)-Tag fusions from exogenously expressed Ub-BiP(19-N)-Tag and Ub-R-BiP(19-N)-Tag, but not Ub-V-BiP(20-N)-Tag, constructs. In ATE1-deficient cells, the target R-BiP(19-651)-GFP band was detected only when the cells were tranfected to express Ub-R-BiP(19-651)-GFP, but not Ub-BiP(19-651)-GF (Cha-Molstad, H., et al. (2015). Nat. Cell Biol. 17(7):917-929).

Western Blotting Analysis: A representative lot detected BiP (GRP78) Nt-Glu19 arginylation induction upon arginine-tRNA-protein transferase 1 (ATE1) 1A7A isoform overexpression or transfection of various dsDNAs, including poly(dA:dT), in HeLa cells. Combined proteasome inhibition and ER stress induction by an 18-hr 10 µM MG132 and 100 nM thapsigargin treatment synergized the two drugs′ efficacy toward cellular Calreticulin Nt-Glu18 arginylation induction (Cha-Molstad, H., et al. (2015). Nat. Cell Biol. 17(7):917-929).

Quality

Evaluated by Western Blotting in HEK293 cell lysate.

Western Blotting Analysis: 1 µg/mL of this antibody detected BiP (GRP78) Nt-Glu19 arginylation induction in 7.5 µg of lysate from (17-hr 3 µM MG132 and 200 nM thapsigargin) treated HEK293 cells.

Target description

~72 kDa observed. 70.62/70.63/70.62/70.63 kDa (bovine/human/mouse/rat) calculated. Uncharacterized bands may be observed in some lysate(s).

Physical form

Affinity purified.

Purified rabbit polyclonal antibody in buffer containing PBS with 0.05% sodium azide.

Storage and Stability

Stable for 1 year at 2-8°C from date of receipt.

Other Notes

Concentration: Please refer to lot specific datasheet.

Disclaimer

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

biological source: rabbit. Quality Level: 100. antibody form: affinity isolated antibody. antibody product type: primary antibodies. clone: polyclonal. purified by: affinity chromatography. species reactivity: human, mouse. species reactivity (predicted by homology): rat (based on 100% sequence homology), bovine (based on 100% sequence homology), nonhuman primates (based on 100% sequence homology). technique(s): ELISA: suitable, dot blot: suitable, immunocytochemistry: suitable, western blot: suitable. NCBI accession no.: NP_005338. UniProt accession no.: P11021. shipped in: ambient. target post-translational modification: unmodified. Gene Information: human ... HSPA5(3309). Storage Class Code: 12 - Non Combustible Liquids. WGK: WGK 2. Flash Point(F): Not applicable. Flash Point(C): Not applicable.