HSP/Chaperone Antibody Kit
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Catalog# K2007
HSP/Chaperone Antibody Kit
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WB
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Human
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Mouse
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Rat
General Overview
Kit Components
| Product Includes | Specification | Application | Reactivity | Mw |
|---|---|---|---|---|
| HSPA14[ET1612-93] | 20μl | WB,IF-Cell,IF-Tissue,IHC-P | Human,Mouse,Rat,Monkey | Predicted band size: 55 kDa |
| Hsp70[ET1601-11] | 20μl | WB,IF-Cell,IF-Tissue,IHC-P,FC | Human,Mouse,Rat | Predicted band size: 70 kDa |
| GRP78 / BIP[HA601076] | 20μl | WB,IHC-P,IF-Cell | Human,Mouse,Rat | Predicted band size: 72 kDa |
| Hsp40[HA723426] | 20μl | WB,IF-Cell,IHC-P,FC | Human,Mouse,Rat,Monkey | Predicted band size: 38 kDa |
| Hsp90 beta[ET1605-56] | 20μl | WB,IF-Cell,IF-Tissue,IHC-P,IP,FC | Human,Mouse,Rat | Predicted band size: 83 kDa |
| Hsp90 alpha[ET1605-57] | 20μl | WB,IF-Cell,IF-Tissue,IHC-P | Human,Mouse,Rat,Monkey | Predicted band size: 85 kDa |
| Calmegin[ET7111-36] | 20μl | WB,IHC-P,FC | Human,Mouse,Rat | 70 kDa |
| P4HB[ET7110-92] | 20μl | WB,IF-Cell,IHC-P,FC | Human,Mouse,Rat,Monkey | Predicted band size: 57 kDa |
| Alpaca anti-Rabbit IgG Fc, Recombinant VHH[HA1031] | 100μl | IP,ELISA,IHC-P,WB | Rabbit | |
| Goat Anti-Mouse IgG (H+L)[HA1006] | 100μl | WB,ELISA,IHC-P | Mouse |
Product Description
The HSP/Chaperone Sampler Kit provides an economical means to investigate protein folding within the cell. The kit contains enough primary and secondary antibodies to perform two Western blot experiments with each antibody.
Product Features
Storage Buffer
1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Storage Instructions
Store at +4℃ after thawing. Aliquot store at -20℃. Avoid repeated freeze / thaw cycles.
Background
HSP70 and HSP90 are molecular chaperones expressed constitutively under normal conditions to maintain protein homeostasis and are induced upon environmental stress . HSP70 and HSP90 interact with unfolded proteins to prevent irreversible aggregation and catalyze the refolding of their substrates in an ATP-dependent manner.</br>HSP40 family proteins bind unfolded proteins and prevent their aggregation, and deliver unfolded protiens to HSP70. HSP60 has primarily been known as a mitochondrial protein that is important for folding key proteins after import into the mitochondria. Calenxin is a calcium-binding protein embedded in the ER membrane that retains newly synthesized glycoproteins inside the ER to ensure proper folding and quality control.Heat shock gene transcription is regulated by a familly of heat shock factors (HSFs), transcriptional activators that bind to heat shock response elements (HSEs) located upstream of all heat shock genes. During attenuation from the heat shock response, HSF1 is repressed by direct binding of HSP70, HSP40/Hdj-1 and HSF binding protein 1 (HSBP1).
Data Links
Background References
1. Nollen, E.A. and Morimoto, R.I. (2002) J. Cell Sci. 115, 2809-2816.
2. Fan, C.Y. et al. (2003) Cell Stress Chaperones 8, 309-316.
3. Jindal, S. et al. (1989) Mol Cell Biol 9, 2279-83.
4. Itoh, H. et al. (2002) Eur. J. Biochem. 269, 5931-5938.
5. Gupta, S. and Knowlton, A.A. J. Cell Mol. Med. 9, 51-58.
6. Deocaris, C.C. et al. (2006) Cell Stress Chaperones 11, 116-128.
7. Lai, H.C. et al. (2007) Am. J. Physiol. Endocrinol. Metab. 292, E292-E297.
8. Bergeron, J.J. et al. (1994) Trends Biochem. Sci. 19, 124-128.
9. Williams, D.B. (2006) J. Cell Sci. 119, 615-623.
10. Kohno, K. et al. (1993) Mol. Cell. Biol. 13, 877-890.
11. Ellgaard, L. and Ruddock, L.W. (2005) EMBO Rep. 6, 28-32.
12. Morimoto, R.I. (1998) Genes Dev. 12, 3788-3796.
13. Satyal, S.H. et al. (1998) Genes Dev. 12, 1962-1974.
Images
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Western blot analysis of Hsp70 on different lysates with Rabbit anti-Hsp70 antibody (ET1601-11) at 1/2,000 dilution.
Lane 1: HeLa cell lysate
Lane 2: A549 cell lysate
Lane 3: MCF7 cell lysate
Lane 4: HCT 116 cell lysate
Lane 5: Mouse brain tissue lysate
Lane 6: Mouse testis tissue lysate
Lane 7: Rat testis tissue lysate
Lysates/proteins at 15 µg/Lane.
Predicted band size: 70 kDa
Observed band size: 70 kDa
Exposure time: Lane 1-4: 43 seconds; Lane 5-7: 2 minutes;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1601-11) at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:50,000 dilution was used for 1 hour at room temperature. -
Western blot analysis of GRP78 / BIP on different lysates with Mouse anti-GRP78 / BIP antibody (HA601076) at 1/1,000 dilution.
Lane 1: MCF7 cell lysate (15 µg/Lane)
Lane 2: HepG2 cell lysate (15 µg/Lane)
Lane 3: Mouse brain tissue lysate (30 µg/Lane)
Lane 4: U-87 MG cell lysate (30 µg/Lane)
Lane 5: RAW264.7 cell lysate (30 µg/Lane)
Lane 6: RAW264.7 treated with 300nM Thapsigargin for 18 hours cell lysate (30 µg/Lane)
Lane 7: Mouse liver tissue lysate (30 µg/Lane)
Lane 8: Rat liver tissue lysate (30 µg/Lane)
Lane 9: Rat pancreas tissue lysate (30 µg/Lane)
Predicted band size: 72 kDa
Observed band size: 72 kDa
Exposure time: Lane 1-3: 11 seconds; Lane 4-9: 4 seconds; ECL: K1801;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA601076) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1/50,000 dilution was used for 1 hour at room temperature. -
Western blot analysis of Hsp90 beta on different lysates with Rabbit anti-Hsp90 beta antibody (ET1605-56) at 1/10,000 dilution.
Lane 1: HEK-293 cell lysate (20 µg/Lane)
Lane 2: HeLa cell lysate (20 µg/Lane)
Lane 3: A431 cell lysate (20 µg/Lane)
Lane 4: K-562 cell lysate (20 µg/Lane)
Lane 5: RAW264.7 cell lysate (20 µg/Lane)
Lane 6: PC-12 cell lysate (20 µg/Lane)
Lane 7: Mouse heart tissue lysate (40 µg/Lane)
Lane 8: Rat heart tissue lysate (40 µg/Lane)
Lane 9: Human liver tissue lysate (40 µg/Lane)
Lane 10: Mouse liver tissue lysate (40 µg/Lane)
Lane 11: Rat liver tissue lysate (40 µg/Lane)
Lane 12: Human kidney tissue lysate (40 µg/Lane)
Lane 13: Mouse kidney tissue lysate (40 µg/Lane)
Lane 14: Rat kidney tissue lysate (40 µg/Lane)
Predicted band size: 83 kDa
Observed band size: 90 kDa
Exposure time: 1 minute 21 seconds;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1605-56) at 1/10,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature. -
Western blot analysis of Hsp90 alpha on different lysates with Rabbit anti-Hsp90 alpha antibody (ET1605-57) at 1/1,000 dilution.
Lane 1: HeLa cell lysate (15 µg/Lane)
Lane 2: A549 cell lysate (15 µg/Lane)
Lane 3: COS-1 cell lysate (15 µg/Lane)
Lane 4: NIH/3T3 cell lysate (15 µg/Lane)
Lane 5: PC-12 cell lysate (15 µg/Lane)
Lane 6: Mouse testis tissue lysate (20 µg/Lane)
Lane 7: Rat testis tissue lysate (20 µg/Lane)
Predicted band size: 85 kDa
Observed band size: 90 kDa
Exposure time: 20 seconds; ECL: K1801;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1605-57) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature. -
Immunohistochemical analysis of paraffin-embedded human kidney tissue with Rabbit anti-HSPA14 antibody (ET1612-93) at 1/200 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1612-93) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX. -
Immunohistochemical analysis of paraffin-embedded human kidney tissue with Rabbit anti-HSPA14 antibody (ET1612-93) at 1/200 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1612-93) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX. -
Immunocytochemistry analysis of NIH/3T3 cells labeling Hsp70 with Rabbit anti-Hsp70 antibody (ET1601-11) at 1/100 dilution.
Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Hsp70 antibody (ET1601-11) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution. -
Immunocytochemistry analysis of HeLa cells labeling Hsp90 beta with Rabbit anti-Hsp90 beta antibody (ET1605-56) at 1/1,000 dilution.
Cells were fixed in 100% precooled methanol for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Hsp90 beta antibody (ET1605-56) at 1/1,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution. -
Immunocytochemistry analysis of NIH/3T3 cells labeling P4HB with Rabbit anti-P4HB antibody (ET7110-92) at 1/250 dilution.
Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-P4HB antibody (ET7110-92) at 1/250 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
Beta tubulin (HA601187, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution. -
Flow cytometric analysis of HepG2 cells labeling P4HB.
Cells were fixed and permeabilized. Then stained with the primary antibody (ET7110-92, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
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