Anti-Cyclin D2 antibody [EPR27468-62] - BSA and Azide free

• WB

Supplier Data

Western blot - Anti-Cyclin D2 antibody [EPR27468-62] - BSA and Azide free (AB308259)

This data was developed using 308258, the same antibody clone in a different buffer formulation.  Blocking and diluting buffer and concentration : Intercept® (TBS) Blocking Buffer diluted with an equal volume of TBS. Lysates at 20 µg per lane. The samples were run on a Bis-Tris gel under reducing conditions. Western blot : Anti-Cyclin D2 antibody (ab308258) staining at 1/1000 dilution, shown in green; Mouse anti-Tubulin antibody DM1A (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab308258 was shown to bind specifically to  Cyclin D2. Target of interest was observed at 31 kDa in wild-type 293T cell lysates (lane 1) with no signal observed at this size in Cyclin D2 knockout cell line ab267318(Cyclin D2 knockout cell lysate ab257875) (lane 2).  To generate this image, samples were first run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane.  Membranes were blocked in a fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4 °C.  Blots were washed in TBS-T, incubated with secondary antibodies Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution for 1 h at room temperature, washed again then imaged.

All lanes:

Western blot - Anti-Cyclin D2 antibody [EPR27468-62] (<a href='/en-us/products/primary-antibodies/cyclin-d2-antibody-epr27468-62-ab308258'>ab308258</a>) at 1/1000 dilution

Lane 1:

Wild-type 293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

CCND2 knockout 293T whole cell lysate at 20 µg

Lane 3:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 4:

C2C12 (mouse myoblast) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (800CW) and Goat Anti-Mouse IgG H&L (680RD) at 1/10000 dilution

Predicted band size: 32 kDa

Observed band size: 31 kDa

false

• WB

Supplier Data

Western blot - Anti-Cyclin D2 antibody [EPR27468-62] - BSA and Azide free (AB308259)

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

ab181602 was used as a GAPDH loading control.

We recommend using a higher sensitive ECL substrate to increase the band intensity.

This data was developed using ab308258, the same antibody clone in a different buffer formulation.

All lanes:

Western blot - Anti-Cyclin D2 antibody [EPR27468-62] (<a href='/en-us/products/primary-antibodies/cyclin-d2-antibody-epr27468-62-ab308258'>ab308258</a>) at 1/1000 dilution

Lane 1:

293T (Human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

U-2 OS (Human bone osteosarcoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

Human heart tissue lysate at 20 µg

Lane 4:

Human thyroid tissue lysate at 20 µg

Lane 5:

Mouse heart tissue lysate at 20 µg

Lane 6:

Mouse thyroid tissue lysate at 20 µg

Lane 7:

Rat heart tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 33 kDa

Observed band size: 31 kDa

true

Exposure time: 40s

• WB

Supplier Data

Western blot - Anti-Cyclin D2 antibody [EPR27468-62] - BSA and Azide free (AB308259)

This data was developed using 308258, the same antibody clone in a different buffer formulation.  Blocking and diluting buffer and concentration : 5% NFDM/TBST. In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution. Exposure time : 180 seconds

All lanes:

Western blot - Anti-Cyclin D2 antibody [EPR27468-62] (<a href='/en-us/products/primary-antibodies/cyclin-d2-antibody-epr27468-62-ab308258'>ab308258</a>) at 1/1000 dilution

Lane 1:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

RAW 264.7 (mouse elson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

Mouse spleen tissue lysate at 20 µg

Lane 5:

Rat spleen tissue 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 1/100000 dilution

Predicted band size: 32 kDa

Observed band size: 31 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-Cyclin D2 antibody [EPR27468-62] - BSA and Azide free (AB308259)

This data was developed using 308258, the same antibody clone in a different buffer formulation. Blocking and diluting buffer and concentration : 5% NFDM/TBST. This antibody does not cross-react with mouse CCND3 and CCND1. In Western blot, anti-His antibody (ab213204) staining at 1/5000 dilution.#8 seconds Exposure time : 8 seconds

All lanes:

Western blot - Anti-Cyclin D2 antibody [EPR27468-62] (<a href='/en-us/products/primary-antibodies/cyclin-d2-antibody-epr27468-62-ab308258'>ab308258</a>) at 1/1000 dilution

Lane 1:

His-tagged mouse CCND2 recombinant protein fragment at 10 ng

Lane 2:

His-tagged mouse CCND3 recombinant protein at 10 ng

Lane 3:

MYC/DDK -tagged mouse CCND1 recombinant protein at 10 ng

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 1/100000 dilution

Predicted band size: 32 kDa

Observed band size: 31 kDa

false

Exposure time: 8s