Anti-SKP2 antibody [EPR3305(2)]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SKP2 antibody [EPR3305(2)] (AB183039)

immunohistochemical analysis of paraffin embedded Human kidney clear cell carcinoma tissue labeling SKP2 with ab183039 at 1/50 followed by secondary staining with Ready to use HRP Polymer for Rabbit IgG and counterstained with Hematoxylin.

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-SKP2 antibody [EPR3305(2)] (AB183039)

Intracellular flow cytometric analysis of 2% paraformaldehyde fixedHeLa cells labeling SKP2 with ab183039 at 1/70 followed byGoat anti rabbit IgG (FITC) at 1/150.Rabbit monoclonal IgG was used asIsotype control.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-SKP2 antibody [EPR3305(2)] (AB183039)

Immunofluorescenct analysis of 4% paraformaldehyde fixed 293T cells labeling SKP2 with ab183039 at 1/100 followed by Goat anti rabbit IgG(Alexa Fluor® 488) at 1/200 (green). Cells were counter stained with Dapi (blue).

• WB

Lab

Western blot - Anti-SKP2 antibody [EPR3305(2)] (AB183039)

Lanes 1 - 4 : Merged signal (red and green). Green - ab183039 observed at 52 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab183039 was shown to recognize in wild-type HAP1 cells as signal was lost at the expected MW in SKP2 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and SKP2 knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% NF Milk. ab183039 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/200 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-SKP2 antibody [EPR3305(2)] (ab183039) at 1/200 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

SKP2 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa whole cell lysate at 20 µg

Lane 4:

MCF7 whole cell lysate at 20 µg

Predicted band size: 45 kDa,47 kDa,55 kDa

Observed band size: 45 kDa,55 kDa

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• WB

Lab

Western blot - Anti-SKP2 antibody [EPR3305(2)] (AB183039)

We recommend to increase the amount of samples or decrease antibody dilution to get clear bands.

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

All lanes:

Western blot - Anti-SKP2 antibody [EPR3305(2)] (ab183039) at 1/200 dilution

Lane 1:

NCCIT (Human pluripotent embryonic carcinoma epithelial cell) whole cell lysates at 20 µg

Lane 2:

HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysates at 20 µg

Lane 3:

MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysates at 20 µg

Lane 4:

Jurkat (Human T cell leukemia T lymphocyte) whole cell lysates at 20 µg

Lane 5:

MDA-MB-231 (Human breast adenocarcinoma epithelial cell) whole cell lysates at 20 µg

Lane 6:

A549 (Human lung carcinoma epithelial cell) whole cell lysates at 20 µg

Lane 7:

F9 (Mouse embryonal carcinoma epithelial cell) whole cell lysates at 20 µg

Lane 8:

NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates at 20 µg

Lane 9:

C6 (Rat glial tumor glial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 47 kDa

false

Exposure time: 180s