VPS35 Recombinant Rabbit Monoclonal Antibody [PSH16-54]
cat.: HA751603
Product Type: Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity: Human, Mouse, Rat, Monkey
Applications: WB, IF-Cell, FC, IHC-P, IP
Clonality: Monoclonal
Clone number: PSH16-54
Form: Liquid
Storage condition: Store at +4℃ after thawing. Aliquot store at -20℃. Avoid repeated freeze / thaw cycles.
Storage buffer: 1*PBS (pH7.4).
Concentration: 1ug/ul
Purification: Protein A affinity purified.
Molecular weight: Predicted band size: 92 kDa
Isotype: IgG
Immunogen: Synthetic peptide within human VSP35 aa 760-795.
Positive control: SH-SY5Y cell lysate, A549 cell lysate, U-937 cell lysate, COS-1 cell lysate, Neuro-2a cell lysate, NIH/3T3 cell lysate, C2C12 cell lysate, C6 cell lysate, NR8383 cell lysate, PC-12 cell lysate, Mouse brain tissue lysate, Mouse colon tissue lysate, Rat brain tissue lysate, Rat colon tissue lysate, SH-SY5Y, Neuro-2a, C6, human brain tissue, human colon carcinoma tissue, human kidney tissue, mouse brain tissue, mouse kidney tissue, rat brain tissue, rat kidney tissue.
Subcellular location: Cytoplasm, Membrane, Endosome, Early endosome, Late endosome.
Recommended Dilutions:
  WB
  IF-Cell
  IHC-P
  FC
  IP
1:5,000
1:100
1:200-1:1,000
1:1,000
1-2μg/sample
Uniprot #: SwissProt: Q96QK1 Human | Q9EQH3 Mouse | G3V8A5 Rat | I0FFY6 Monkey
Alternative names: DKFZp434E1211 DKFZp434P1672 FLJ10752 FLJ13588 FLJ20388 hVPS35 Maternal embryonic 3 Maternal-embryonic 3 MEM 3 MEM3 PARK17 TCCCTA00141 Vacuolar protein sorting 35 Vacuolar protein sorting 35 homolog Vacuolar protein sorting associated protein 35 Vacuolar protein sorting-associated protein 35 Vesicle protein sorting 35 VPS 35
Images
HA751603_1.jpg Fig1: Western blot analysis of VPS35 on different lysates with Rabbit anti-VPS35 antibody (HA751603) at 1/5,000 dilution.

Lane 1: SH-SY5Y cell lysate (20 µg/Lane)
Lane 2: A549 cell lysate (20 µg/Lane)
Lane 3: U-937 cell lysate (20 µg/Lane)
Lane 4: COS-1 cell lysate (20 µg/Lane)
Lane 5: Neuro-2a cell lysate (20 µg/Lane)
Lane 6: NIH/3T3 cell lysate (20 µg/Lane)
Lane 7: C2C12 cell lysate (20 µg/Lane)
Lane 8: C6 cell lysate (20 µg/Lane)
Lane 9: NR8383 cell lysate (20 µg/Lane)
Lane 10: PC-12 cell lysate (20 µg/Lane)
Lane 11: Mouse brain tissue lysate (40 µg/Lane)
Lane 12: Mouse colon tissue lysate (40 µg/Lane)
Lane 13: Rat brain tissue lysate (40 µg/Lane)
Lane 14: Rat colon tissue lysate (40 µg/Lane)

Predicted band size: 92 kDa
Observed band size: 80 kDa

Exposure time: 59 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 (HA751603) at 1/5,000 dilution was used in primary antibody dilution (K1803) at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
HA751603_2.jpg Fig2: Western blot analysis of VPS35 on different lysates with Rabbit anti-VPS35 antibody (HA751603) at 1/5,000 dilution.

Lane 1: HAP1-parental cell lysate (10 µg/Lane)
Lane 2: HAP1-VPS35 KD cell lysate (10 µg/Lane)

Predicted band size: 92 kDa
Observed band size: 80 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 (HA751603) at 1/5,000 dilution was used in primary antibody dilution (K1803) at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
HA751603_3.jpg Fig3: Immunocytochemistry analysis of SH-SY5Y cells labeling VPS35 with Rabbit anti-VPS35 antibody (HA751603) 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-VPS35 antibody (HA751603) 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.
HA751603_4.jpg Fig4: Immunocytochemistry analysis of Neuro-2a cells labeling VPS35 with Rabbit anti-VPS35 antibody (HA751603) 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-VPS35 antibody (HA751603) 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.
HA751603_5.jpg Fig5: Immunocytochemistry analysis of C6 cells labeling VPS35 with Rabbit anti-VPS35 antibody (HA751603) 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-VPS35 antibody (HA751603) 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.
HA751603_6.jpg Fig6: Immunohistochemical analysis of paraffin-embedded human brain tissue with Rabbit anti-VPS35 antibody (HA751603) 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 (HA751603) 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.
HA751603_7.jpg Fig7: Immunohistochemical analysis of paraffin-embedded human colon carcinoma tissue with Rabbit anti-VPS35 antibody (HA751603) 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 (HA751603) 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.
HA751603_8.jpg Fig8: Immunohistochemical analysis of paraffin-embedded human kidney tissue with Rabbit anti-VPS35 antibody (HA751603) 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 (HA751603) 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.
HA751603_9.jpg Fig9: Immunohistochemical analysis of paraffin-embedded mouse brain tissue with Rabbit anti-VPS35 antibody (HA751603) 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 (HA751603) 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.
HA751603_10.jpg Fig10: Immunohistochemical analysis of paraffin-embedded mouse kidney tissue with Rabbit anti-VPS35 antibody (HA751603) 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 (HA751603) 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.
HA751603_11.jpg Fig11: Immunohistochemical analysis of paraffin-embedded rat brain tissue with Rabbit anti-VPS35 antibody (HA751603) 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 (HA751603) 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.
HA751603_12.jpg Fig12: Immunohistochemical analysis of paraffin-embedded rat kidney tissue with Rabbit anti-VPS35 antibody (HA751603) 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 (HA751603) 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.
HA751603_13.jpg Fig13: Flow cytometric analysis of SH-SY5Y cells labeling VPS35.

Cells were fixed and permeabilized. Then stained with the primary antibody (HA751603, 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).
HA751603_14.jpg Fig14: Flow cytometric analysis of Neuro-2a cells labeling VPS35.

Cells were fixed and permeabilized. Then stained with the primary antibody (HA751603, 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).
HA751603_15.jpg Fig15: Flow cytometric analysis of C6 cells labeling VPS35.

Cells were fixed and permeabilized. Then stained with the primary antibody (HA751603, 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).
HA751603_16.jpg Fig16: VPS35 was immunoprecipitated from 0.2 mg A549 cell lysate with HA751603 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA751603 at 1/5,000 dilution. HRP Conjugated Anti-Rabbit IgG for IP Nano-secondary antibody at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: A549 cell lysate (input)
Lane 2: HA751603 IP in A549 cell lysate
Lane 3: Rabbit IgG instead of HA751603 in A549 cell lysate

Blocking/Dilution buffer: primary antibody dilution (K1803)
Exposure time: 3 minutes; ECL: K1801
HA751603_17.jpg Fig17: VPS35 was immunoprecipitated from 0.2 mg NIH/3T3 cell lysate with HA751603 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA751603 at 1/5,000 dilution. HRP Conjugated Anti-Rabbit IgG for IP Nano-secondary antibody at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: NIH/3T3 cell lysate (input)
Lane 2: HA751603 IP in NIH/3T3 cell lysate
Lane 3: Rabbit IgG instead of HA751603 in NIH/3T3 cell lysate

Blocking/Dilution buffer: primary antibody dilution (K1803)
Exposure time: 46 seconds; ECL: K1801
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