HAK21186

Metabolic-Exosome Pathway Antibody Sampler Kit

cat.: HAK21186

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.
Concentration:	1ug/ul
Uniprot #:	SwissProt: Q86YN6 Human \| Q9UBK2 Human \| 415302 Mouse \| O70343 Mouse \| Q8VHJ7 Mouse \| Q811R2 Rat \| Q9QYK2 Rat \| P21926 Human \| P40240 Mouse \| P40241 Rat \| P17813 Human \| Q99816 Human \| Q61187 Mouse \| Q6IRE4 Rat \| Q13131 Human \| Q5EG47 Mouse \| P54645 Rat \| Q13131 Human \| Q5EG47 Mouse \| P16220 Human \| Q01147 Mouse \| P15337 Rat \| P16220 Human \| Q01147 Mouse \| P15337 Rat

Images

Fig1: Western blot analysis of PGC1 alpha+beta on different lysates with Rabbit anti-PGC1 alpha+beta antibody (ET1702-96) at 1/2,000 dilution.

Lane 1: SK-Br-3 cell lysate
Lane 2: NIH/3T3 cell lysate
Lane 3: C6 cell lysate
Lane 4: Human kidney tissue lysate
Lane 5: Mouse kidney tissue lysate
Lane 6: Rat kidney tissue lysate
Lane 7: Mouse brain tissue lysate
Lane 8: Rat brain tissue lysate
Lane 9: Mouse heart tissue lysate
Lane 10: Rat heart tissue lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 91/113 kDa
Observed band size: 91/113 kDa

Exposure time: 43 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 (ET1702-96) at 1/2,000 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.

Fig2: Immunohistochemical analysis of paraffin-embedded human kidney tissue with Rabbit anti-CD9 antibody (ET1601-9) 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 (ET1601-9) 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.

Fig3: Western blot analysis of CD105 on different lysates with Rabbit anti-CD105 antibody (HA720072) at 1/1,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: HeLa cell lysate treated with deglycosylation
Lane 3: HUVEC cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 71 kDa
Observed band size: 71-100 kDa

Exposure time: 1 minute; 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 (HA720072) 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: Immunohistochemical analysis of paraffin-embedded human spleen tissue with Rabbit anti-CD105 antibody (HA720072) 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 (HA720072) 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.

Fig5: Application: IF-Tissue

Species: Human

Site: Spleen

Sample: Paraffin-embedded section

Antibody concentration: 1/200

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; 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 (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: Western blot analysis of TSG101 with anti-TSG101 antibody[JJ0900] (ET1701-59) at 1/2,000 dilution.
Lane 1: Wild-type Hela whole cell lysate (10 µg).
Lane 2/3: TSG101 knockdown Hela whole cell lysate (10 µg).

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary antibody (ET1701-59, 1/2,000) was used in 5% BSA 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.

Fig8: 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.

Fig9: Western blot analysis of AMPK alpha 1 on different lysates with Rabbit anti-AMPK alpha 1 antibody (ET1608-40) at 1/5,000 dilution and competitor's antibody at 1/2,000 dilution.

Lane 1: HeLa cell lysate (20 µg/Lane)
Lane 2: MCF7 cell lysate (20 µg/Lane)
Lane 3: K-562 cell lysate (20 µg/Lane)
Lane 4: 293T cell lysate (20 µg/Lane)
Lane 5: HT-29 cell lysate (20 µg/Lane)
Lane 6: L-929 cell lysate (20 µg/Lane)
Lane 7: C6 cell lysate (20 µg/Lane)

Predicted band size: 64 kDa
Observed band size: 64 kDa

Exposure time: 1 minute 10 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 (ET1608-40) 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.

Fig10: Immunocytochemistry analysis of HeLa cells labeling AMPK alpha 1 with Rabbit anti-AMPK alpha 1 antibody (ET1608-40) at 1/250 dilution and competitor's antibody at 1/250 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-AMPK alpha 1 antibody (ET1608-40) at 1/250 dilution and competitor's antibody 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 (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.

Fig11: Western blot analysis of Phospho-AMPK alpha 1(S496) on HUVEC cell lysates.

Lane 1: HUVEC cells, whole cell lysate, 10ug/lane
Lane 2/3: HUVEC cells treated with UV(2 hours recovery), whole cell lysates, 10ug/lane
Lane 4: HUVEC cells treated with UV(2 hours recovery), then treated with 2.8ug/ul lambda-PP for 30 minutes, whole cell lysates, 10ug/lane

All lanes :
Anti-Phospho-AMPK alpha 1(S496) antibody (ET1612-72) at 1:500 dilution. Anti-GAPDH antibody (ET1601-4) at 1:10,000 dilution. Goat Anti-Rabbit IgG H&L (HRP) (HA1001) at 1/200,000 dilution.

Predicted band size:64 kDa
Observed band size:64 kDa

Blocking and diluting buffer: 5% BSA.

Exposure time: 2 minutes 14 seconds

Fig12: Immunocytochemistry analysis of HeLa cells treated with or without Lambda Protein Phosphatase for 1 hour labeling Phospho-Creb (S133) with Rabbit anti-Phospho-Creb (S133) antibody (ET7107-93) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 10 minutes at 37 ℃, permeabilized with 0.05% Triton X-100 in PBS for 20 minutes, and then blocked with 2% negative goat serum for 30 minutes at room temperature. Cells were then incubated with Rabbit anti-Phospho-Creb (S133) antibody (ET7107-93) at 1/100 dilution in 2% negative goat serum overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. 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: Immunocytochemistry analysis of NIH/3T3 cells labeling Phospho-Creb (S133) with Rabbit anti-Phospho-Creb (S133) antibody (ET7107-93) 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-Phospho-Creb (S133) antibody (ET7107-93) 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.

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