Chaperone-Mediated Autophagy Antibody Sampler Kit
cat.: HAK21185
Product Type: Antibody Sampler Kit
Species reactivity: Human
Applications: WB, IHC-P
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.
Concentration: 1ug/ul
Uniprot #: SwissProt: P13473 Human | P17047 Mouse | P17046 Rat | P13473 Human | P11142 Human | P63017 Mouse | P63018 Rat | P25685 Human | Q9QYJ3 Mouse | P07900 Human | P08238 Human | P07901 Mouse | P11499 Mouse | P34058 Rat | P82995 Rat | P31948 Human | Q60864 Mouse | O35814 Rat
Entrez Gene: 361384 Rat
Images
HAK21185_1.jpg Fig1: Western blot analysis of LAMP2a on different lysates with Rabbit anti-LAMP2a antibody (ET1601-24) at 1/5,000 dilution.

Lane 1: SK-MEL-28 cell lysate (20 µg/Lane)
Lane 2: HeLa cell lysate (20 µg/Lane)
Lane 3: JAR cell lysate (20 µg/Lane)
Lane 4: U-937 cell lysate (20 µg/Lane)
Lane 5: RAW264.7 cell lysate (15 µg/Lane)
Lane 6: NIH/3T3 cell lysate (15 µg/Lane)
Lane 7: PC-12 cell lysate (15 µg/Lane)
Lane 8: Mouse liver tissue lysate (30 µg/Lane)
Lane 9: Rat liver tissue lysate (30 µg/Lane)
Lane 10: Rat lung tissue lysate (30 µg/Lane)

Predicted band size: 45 kDa
Observed band size: 70-140 kDa

Exposure time: Lane 1-4: 2 minutes 37 seconds; Lane 5-10: 5 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 (ET1601-24) at 1/5,000 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.
HAK21185_2.jpg Fig2: LAMP2a was immunoprecipitated from 0.2 mg SK-MEL-28 cell lysate with ET1601-24 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using ET1601-24 at 1/2,000 dilution. HRP Conjugated Rabbit IgG kappa light chain antibody (M1208-2) at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: SK-MEL-28 cell lysate (Input)
Lane 2: ET1601-24 IP in SK-MEL-28 cell lysate
Lane 3: Rabbit IgG instead of ET1601-24 in SK-MEL-28 cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 20 seconds; ECL: K1801
HAK21185_3.jpg Fig3: Immunohistochemical analysis of paraffin-embedded human kidney tissue with Rabbit anti-LAMP2a antibody (ET1601-24) at 1/500 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 (ET1601-24) at 1/500 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.
HAK21185_4.jpg Fig4: Western blot analysis of LAMP2 on different lysates with Mouse anti-LAMP2 antibody (HA601192) at 1/1,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: Jurkat cell lysate
Lane 3: HepG2 cell lysate
Lane 4: HUVEC cell lysate
Lane 5: JAR cell lysate
Lane 6: HEK-293 cell lysate
Lane 7: THP-1 cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 45 kDa
Observed band size: 110 kDa

Exposure time: 3 minute;

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 (HA601192) 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.
HAK21185_5.jpg Fig5: Western blot analysis of LAMP2 on different lysates with Mouse anti-LAMP2 antibody (HA601192) at 1/2,000 dilution.

Lane 1: HAP1-parental cell lysate
Lane 2: HAP1-LAMP2 KD cell lysate

Lysates/proteins at 10 µg/Lane.

Predicted band size: 45 kDa
Observed band size: 110 kDa

Exposure time: 3 minutes; 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 (HA601192) at 1/2,000 dilution was used in primary antibody dilution (K1803) at 4℃ overnight. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1/50,000 dilution was used for 1 hour at room temperature.
HAK21185_6.jpg Fig6: Immunohistochemical analysis of paraffin-embedded human kidney tissue with Mouse anti-LAMP2 antibody (HA601192) at 1/5,000 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 (HA601192) at 1/5,000 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.
HAK21185_7.jpg Fig7: Western blot analysis of Hsc70 on different lysates with Rabbit anti-Hsc70 antibody (ET1602-33) at 1/1,000 dilution.

Lane 1: HeLa cell lysate (20 µg/Lane)
Lane 2: A431 cell lysate (20 µg/Lane)
Lane 3: MCF7 cell lysate (20 µg/Lane)
Lane 4: NIH/3T3 cell lysate (20 µg/Lane)
Lane 5: PC-12 cell lysate (20 µg/Lane)
Lane 6: Mouse brain tissue lysate (40 µg/Lane)
Lane 7: Rat brain tissue lysate (40 µg/Lane)

Predicted band size: 71 kDa
Observed band size: 71 kDa

Exposure time: 2 minutes; 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 (ET1602-33) 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.
HAK21185_8.jpg Fig8: Immunocytochemistry analysis of HEK-293 cells labeling Hsp40 with Rabbit anti-Hsp40 antibody (HA723426) at 1/100 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-Hsp40 antibody (HA723426) 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 (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.
HAK21185_9.jpg Fig9: Western blot analysis of Hsp90 on different lysates with Rabbit anti-Hsp90 antibody (HA722689) at 1/10,000 dilution.

Lane 1: HeLa cell lysate (20 µg/Lane)
Lane 2: A549 cell lysate (20 µg/Lane)
Lane 3: NIH/3T3 cell lysate (20 µg/Lane)
Lane 4: C6 cell lysate (20 µg/Lane)
Lane 5: COS-1 cell lysate (20 µg/Lane)
Lane 6: Mouse testis tissue lysate (40 µg/Lane)
Lane 7: Rat testis tissue lysate (40 µg/Lane)

Predicted band size: 85 kDa
Observed band size: 85 kDa

Exposure time: 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 (HA722689) at 1/10,000 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.
HAK21185_10.jpg Fig10: Immunohistochemical analysis of paraffin-embedded rat testis tissue with Rabbit anti-Hsp90 antibody (HA722689) at 1/1,000 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 (HA722689) at 1/1,000 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.
Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.