HAK21125

Exosome Essentials Antibody Sampler Kit

cat.: HAK21125

Product Type:	Antibody Sampler Kit
Species reactivity:	Human
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: P27824 Human \| P35564 Mouse \| P35565 Rat \| P21926 Human \| P08962 Human \| P60033 Human \| P35762 Mouse \| Q62745 Rat \| P17879 Mouse \| Q61696 Mouse \| P0DMV9 Human \| P0DMV8 Human \| Q07439 Rat \| Q99816 Human \| Q61187 Mouse \| Q6IRE4 Rat

Images

Fig1: Western blot analysis of CD9 on different lysates with Rabbit anti-CD9 antibody (HA721533) at 1/5,000 dilution and competitor's antibody at 1/2,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: K-562 cell lysate (negative)
Lane 3: MCF7 cell lysate
Lane 4: HepG2 cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 25 kDa
Observed band size: 20 kDa

Exposure time: Lane 1-4 (left): 53 seconds; Lane 1-4 (right): 3 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 (HA721533) at 1/5,000 dilution and competitor's antibody at 1/2,000 dilution were 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.

Fig2: Western blot analysis of CD9 on different lysates with Rabbit anti-CD9 antibody (HA721533) at 1/2,000 dilution.

Lane 1: HepG2-si NT cell lysate
Lane 2: HepG2-si CD9 cell lysate

Lysates/proteins at 10 µg/Lane.

Predicted band size: 25 kDa
Observed band size: 20 kDa

Exposure time: 1 minute 55 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 (HA721533) 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.

Fig3: Western blot analysis of CD63 on different lysates with Rabbit anti-CD63 antibody (HA722731) at 1/1,000 dilution.

Lane 1: SK-MEL-28 cell lysate
Lane 2: Jurkat cell lysate (negative)
Lane 3: U-87 MG cell lysate
Lane 4: HUVEC cell lysate
Lane 5: K-562 cell lysate
Lane 6: HEK-293 cell lysate
Lane 7: HL-60 cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 26 kDa
Observed band size: 30-65 kDa
Exposure time: Lane 1: 4 seconds; Lane 2-7: 18 seconds; ECL: K1802;
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 (HA722731) 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.

Fig4: Western blot analysis of TAPA1/CD81 on different lysates with Rabbit anti-TAPA1/CD81 antibody (ET1611-87) at 1/1,000 dilution.

Lane 1: HCT 116-si NT cell lysate
Lane 2: HCT 116-si TAPA1/CD81 cell lysate

Lysates/proteins at 20 µg/Lane.
Predicted band size: 26 kDa
Observed band size: 25 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 (ET1611-87) 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.

Fig5: 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.

Fig6: Western blot analysis of TSG101 on different lysates with Rabbit anti-TSG101 antibody (ET1701-59) at 1/2,000 dilution and competitor's antibody at 1/1,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: K-562 cell lysate
Lane 3: MCF7 cell lysate
Lane 4: Jurkat cell lysate
Lane 5: C2C12 cell lysate
Lane 6: PC-12 cell lysate

Lysates/proteins at 20 µg/Lane.
Predicted band size: 44 kDa
Observed band size: 44/47 kDa
Exposure time: 3 minutes 20 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 (ET1701-59) at 1/2,000 dilution and competitor's antibody at 1/1,000 dilution were 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.

	Fig7: Immunohistochemical analysis of paraffin-embedded human kidney tissue with Rabbit anti-Calnexin antibody (ET1611-86) 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 (ET1611-86) 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.
	Fig8: Immunohistochemical analysis of paraffin-embedded human lung carcinoma tissue with Rabbit anti-CD9 antibody (HA721533) 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 (HA721533) 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.
	Fig9: Calnexin was immunoprecipitated from 0.2 mg HeLa cell lysate with ET1611-86 at 2 µg/25 µl agarose. Western blot was performed from the immunoprecipitate using ET1611-86 at 1/1,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature. Lane 1: HeLa cell lysate (input) Lane 2: ET1611-86 IP in HeLa cell lysate Lane 3: Rabbit IgG instead of ET1611-86 in HeLa cell lysate Blocking/Dilution buffer: 5% NFDM/TBST Exposure time: 1 minute 5 seconds; ECL: K1801

Fig10: CD63 was immunoprecipitated from 0.2 mg SK-MEL-28 cell lysate with HA722731 at 2 µg/25 µl agarose. Western blot was performed from the immunoprecipitate using HA722731 at 1/1,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: SK-MEL-28 cell lysate (input)
Lane 2: HA722731 IP in SK-MEL-28 cell lysate
Lane 3: Rabbit IgG instead of HA722731 in SK-MEL-28 cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 1 minute 31 seconds; ECL: K1801

Fig11: Immunocytochemistry analysis of A549 cells labeling Hsp70 with Rabbit anti-Hsp70 antibody (ET1601-11) 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-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.

Fig12: 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.

Fig13: Flow cytometric analysis of A549 cells labeling Hsp70.

Cells were fixed and permeabilized. Then stained with the primary antibody (ET1601-11, 1/100) (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).

Fig14: Immunocytochemistry analysis of HeLa cells labeling TSG101 with Rabbit anti-TSG101 antibody (ET1701-59) at 1/200 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-TSG101 antibody (ET1701-59) at 1/200 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.

Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.