PA1-014A

antibody from Invitrogen Antibodies

Targeting: HSPA5 BiP, GRP78

Provider product page for PA1-014A

Western blot

Immunocytochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [29]
Comments [0]
Validations
Western blot [2]
Immunocytochemistry [6]
Other assay [14]

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Product number: PA1-014A - Provider product page
Provider: Invitrogen Antibodies
Product name: GRP78 Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: PA1-014A detects glucose regulated protein/BiP (GRP78) human, rat, mouse, and canine samples. PA1-014A has been successfully used in Western blot and immunofluorescence procedures. By Western blot, this antibody detects a 78 kDa protein representing GRP78 from rat liver lysates. The PA1-014A immunogen is a synthetic peptide corresponding to residues C T(643) G E E D T S E K D E L(654) of rat GRP78. Reconstitute with PBS.
Reactivity: Human, Mouse, Rat, Canine
Host: Rabbit
Isotype: IgG
Vial size: 100 µg
Concentration: 1 mg/mL
Storage: -20° C, Avoid Freeze/Thaw Cycles

HSPA5 protein structure - PA1-014A shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Glucose Regulated Protein 78 using anti-Glucose Regulated Protein 78 polyclonal antibody (Product # PA1-014A) shows staining in p19 Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Glucose Regulated Protein 78 using anti-Glucose Regulated Protein 78 polyclonal antibody (Product # PA1-014A) shows staining in HMVEC Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Glucose Regulated Protein 78 using anti-Glucose Regulated Protein 78 polyclonal antibody (Product # PA1-014A) shows staining in p19 Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Grp78/BiP (green) in MDCK cells. The cells were permeabilized with 0.1% Triton X-100 in PBS for 15 minutes, and blocked with 3% BSA in PBS (Product # 37525) for 15 minutes at room temperature. Cells were stained with a Grp78/BiP rabbit polyclonal antibody (Product # PA1-014A), at a concentration of 10 µg/mL in blocking buffer for at least 1 hour at room temperature, and then incubated with a Goat anti-rabbit IgG Superclonal secondary antibody, Alexa Fluor 488 conjugate (Product # A27034) at a dilution of 1:1000 for 30 minutes at room temperature (green). Nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ToxInsight Instrument at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Glucose Regulated Protein 78 using anti-Glucose Regulated Protein 78 polyclonal antibody (Product # PA1-014A) shows staining in Hela Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of GRP78 was performed using 70% confluent log phase MCF7 cells and MCF7 cells treated with 1 µM of Thapsigargin for 24 hours. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with GRP78 Rabbit Polyclonal Antibody (Product # PA1-014A) at 5 µg/mL in 0.1% BSA, incubated at 4 degree Celsius overnight and then with Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, Alexa Fluor 488 (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing strong Cytoplasmic localization. Panel e shows untreated cells with weak cytoplasmic signal. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 7 ER stress response evaluation. Immunoblot of Grp78 and actin as loading control in whole cell lysates from cells growing in standard conditions (line 1, WT cells in control medium; line 2, CIP-R cells in medium added by 0.2 mM ciprofloxacin), or in WT cells exposed for 24 h to 0.2 mM ciprofloxacin (line 3), or in cells exposed for 24 h to 0.5 ug/ml tunicamycin (TM; line 4, WT cells; line 5, CIP-R cells). The anti-Grp78 is a rabbit antiserum used at 1:500 dilution. The hatched square highlights the standard conditions of culture for each cell line.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 Effect of miR-424 on UPR in HEK 293T cells. ( A ) Upper panel, shows a schematic representation of lentiviral vector used to generate miR-424 expressing clones. Lower panel, expression of GFP was monitored in 293T-control and 293T-miR-424 cells after treatment with (1.0 mug/ml) of doxycycline for 24 hours. ( B ) 293T-control and 293T-miR-424 cells were treated with (1.0 mug/ml) of doxycycline for 24 h and expression levels of miR-424 was quantified by qRT-PCR, normalizing against snoRNA. Error bars represent mean +- S.D. from two independent experiments performed in triplicate. *P < 0.05, two-tailed unpaired t-test comparing the expression in 293T-control and 293T-miR-424 cells. ( C ) 293T-control and 293T-miR-424 were transfected with the indicated UPR pathway reporter gene (ATF6, CHOP, XBP1) and 24 hours post transfection cells were treated with TG (1.0 muM). Normalized Firefly luciferase activities (Firefly/Renilla) relative to untreated control are shown. *P < 0.05, two-tailed unpaired t-test comparing the increase in luciferase activity in 293T-control and 293T-miR-424 cells. ( D ) 293T-control and 293T-miR-424 cells were either untreated or treated with (1.0 muM) TG for indicated time points and immunoblotting of total protein was performed using antibodies against ATF6, spliced XBP1, PERK, GRP78, phospho-eIF2alpha, total eIF2alpha and beta-actin. ( E ) Autorads obtained after probing the membrane with GRP78 antibody were analysed using Image J. Relative intens

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Comparison of gefitinib resistance-related proteins in HCC827 and HCC827GR cells. ( a ) ER stress response elements (GRP78 (glucose regulated protein of a78 kDa molecular weight) and CHOP (CCAAT/enhancer-binding protein homologous protein)), beta-catenin, and cyclooxygenase-2 (COX-2). ( b ) Epithelial (E-cadherin) and mesenchymal (vimentin and N-cadherin) markers, as well as bypass signaling molecules (AXL and p-MET). ( c ) Cancer stem markers (CD133, CD44, and ABCG2). Whole-cell lysates were analyzed by Western blot for the proteins of interest, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. The numbers under the bands show the relative densitometric ratios to the bands of the loading control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 8 Effect of anthocyanins from the flower petals of Hibiscus syriacus L. (Malvaceae, AHs) on ER stress and mtROS production. ( A ) and ( B ) The expression of GRP78, ATF4, p-eIF1alpha, CHOP, and beta-actin protein (left) and relative density (right). ( C ) The staining of MitoSOX Red and MitoTracker Green. ( D ) Survival rate of zebrafish. * p < 0.05 and *** p < 0.001 vs. UVB-irradiated cells and ### p < 0.001 vs. untreated cells (UT).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 6. Effect of purified EDEM1/2/3 on M8B present in mCD3-delta-DeltaTM-HA. ( A ) ( a ) Eluate obtained from EDEM1, 3-double knockout (DKO) cells overexpressing TAP-mCD3-delta-DeltaTM-HA was subjected to SDS-PAGE under reducing conditions, silver-stained, and then analyzed by immunoblotting using anti-HA antibody. ( b ) Eluate in ( a ) was untreated (-) or treated (+) with EndoH, subjected to SDS-PAGE under reducing conditions, and analyzed by immunoblotting using anti-HA, anti-Myc, and anti-GRP78 (which is identical to BiP) antibodies. ( B ) ( a ) Eluate in ( A ) was incubated with purified EDEM1, EDEM2-TXNDC11 complex, or EDEM3(Q543N) for the indicated time, and then analyzed by immunoblotting using anti-Myc antibody. ( b ) N -glycans prepared from samples in ( a ) after 24 hr incubation were analyzed by mass spectrometry (MS). This experiment was conducted once.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 7. Effect of purified EDEM1/2/3 on M8B present in ATF6alpha(C). ( A ) ( a ) Eluate obtained from EDEM1, 3-double knockout (DKO) cells overexpressing ATF6alpha(C)-TAP2 was subjected to SDS-PAGE under reducing conditions, silver-stained, and analyzed by immunoblotting using anti-Myc antibody. ( b ) Eluate in ( a ) was untreated (-) or treated (+) with EndoH, subjected to SDS-PAGE under reducing conditions, and analyzed by immunoblotting using anti-Myc and anti-GRP78 (which is identical to BiP) antibodies. ( B ) ( a ) Eluate in ( A ) was incubated with purified EDEM1, EDEM2-TXNDC11 complex, or EDEM3(Q543N) for the indicated time, and then analyzed by immunoblotting using anti-Myc antibody. ( b ) N -glycans prepared from samples in ( a ) after 24 hr incubation were analyzed by mass spectrometry (MS). This experiment was conducted once.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 7 Irp2 deficiency impairs Fe-S cluster protein function and causes ER stress. a CRISPR/Cas9 depletion of Irp2 in INS-1 832/13 cells (sgIrp2.1, sgIrp2.2) and control parental cells grown in the presence of FAC (10 ug/ml), DFO (50 uM), or no treatment (NT) for 18 h. Irp2, Fth1, Ftl1 and TfR1 were assessed by western blot analysis to show efficacy of Irp2 knockdown and appropriate iron regulation. beta-Actin is a loading control. Asterisk, nonspecific band. b The total iron content measured by ICP-MS and normalized to total cellular protein ( n = 3 independent biological experiments). c , d Mitochondrial ( c ) and cytosolic ( d ) aconitase activity in control, sgIrp2.1, and sgIrp2.2 INS-1 cells grown in medium with or without supplemental FAC and normalized to total cellular protein. e , f Complex I activity ( e ) and complex IV activity ( f ) in lysates from control EV and shIrp2 cells grown in medium with or without supplemental FAC and normalized to total cellular protein ( n >= 3 independent biological experiments). g ATP production in control INS-1, sgIrp2.1, and sgIrp2.2 cells grown in medium with or without supplemental FAC and assayed under basal (5 mM) glucose and after stimulation with 15 mM glucose for 1 h. ATP production was normalized to total cellular protein ( n = 5 independent biological experiments). h Western blot analysis of eIF2alpha-P, eIF2alpha-total, and Grp78/BiP levels in sgIrp2.1 and sgIrp2.2 cells under basal glucose (5 mM) and after stimulation w

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 HRECs exposed to TNF-alpha, and high glucose for 24 h demonstrated increased GRP78 translocation. HRECs silenced for KDELR1 (KDEL-KD), confirmed with protein expression of KDELR1 in total cell lysates ( A ). GRP78 protein expression in cytosolic (Cyto) and membrane (Mem) fractions from various treatments. Na + /K + -ATPase served as an internal control for membrane fraction, while beta-tubulin represented a cytosolic fraction ( B ). Representative confocal immunofluorescence images of the cells stained for GRP78 and WGA ( C ). Data represent Mean +- SEM from 3 independent experiments performed in duplicates. ***p < 0.001; **p < 0.01; *p < 0.05, # p > 0.05.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 7 HRECs exposed to TNF-alpha, and high glucose for 24 h demonstrated alterations in VE-Cadherin and GRP78 association. Whole cell lysate were immunoprecipitated with antibody against VE-Cadherin, and the immunocomplexes were analyzed by immunoblotting for GRP78 ( A ). Whole cell lysates were immunoprecipitated with complex partners (p-120 and beta-catenin) and immunoblotted with GRP78 ( B ). beta-tubulin served as an internal control. Data represent Mean +- SEM from 3 independent experiments performed in duplicates. ***p < 0.001; **p < 0.01.