HSP/Chaperone Antibody Kit
cat.: K2007
Product Type: Antibody Sampler Kit
Species reactivity: Human, Mouse, Rat
Applications: WB
Form: Liquid
Storage condition: Store at +4℃ after thawing. Aliquot store at -20℃. Avoid repeated freeze / thaw cycles.
Storage buffer: 1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Uniprot #: SwissProt: Q0VDF9 Human | Q99M31 Mouse | Q6AYB4 Rat | P17879 Mouse | Q61696 Mouse | P0DMV9 Human | P0DMV8 Human | Q07439 Rat | P11021 Human | P20029 Mouse | P06761 Rat | P08238 Human | P11499 Mouse | P34058 Rat | P07900 Human | P07901 Mouse | P82995 Rat | O14967 Human | P52194 Mouse | P07237 Human | P09103 Mouse | P04785 Rat
Alternative names: Heat shock 70 kDa protein 14 Heat shock 70kDa protein 14 Heat shock protein HSP60 Heat shock protein hsp70 related protein HSP60 HSP70 4 HSP70 like protein 1 HSP70-like protein 1 HSP70L1 HSP7E_HUMAN HSPA 14 HSPA14 MGC131990
Images
K2007_1.jpg Fig1: 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.
K2007_2.jpg Fig2: 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.
K2007_3.jpg Fig3: 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.
K2007_4.jpg Fig4: 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.
K2007_5.jpg Fig5: 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.
K2007_6.jpg Fig6: 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.
K2007_7.jpg Fig7: 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.
K2007_8.jpg Fig8: 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.
K2007_9.jpg Fig9: 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.
K2007_10.jpg Fig10: 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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