Anti-Cyclin D1 antibody [SP4] - BSA and Azide free

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Cyclin D1 antibody [SP4] - BSA and Azide free (AB239794)

ab16663 staining Cyclin D1 in wild-type HAP1 cells (top panel) and CCND1 knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab16663 at 1/250 dilution and ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab16663).

• IHC-P

AbReview19448****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cyclin D1 antibody [SP4] - BSA and Azide free (AB239794)

ab16663 staining Cyclin D1 in Human urinary tract tissue sections by Immunohistochemistry (IHC-P - formaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 10% BSA for 30 minutes at room temperature; antigen retrieval was by heat mediation in citrate buffer. Samples were incubated with primary antibody (1/100 in PBS) for 1 hour. An undiluted HRP-conjugated Goat anti-rabbit IgG polyclonal was used as the secondary antibody.This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab16663).

This image is courtesy of an Abreview submitted by Karin Birkenkamp-Demtroeder

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Cyclin D1 antibody [SP4] - BSA and Azide free (AB239794)

ab16663 staining Cyclin D1 in MCF7 cells. The cells were fixed with 4% formaldehyde (10 min), permeabilized in 0.1% Triton X-100 for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab16663 at a working dilution of 1/250 and ab195889, Mouse monoclonal [DM1A] to alpha Tubulin (Alexa Fluor® 594, shown in red) at 1/250 overnight at +4°C, followed by a further incubation at room temperature for 1h with an anti-rabbit AlexaFluor® 488 (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab16663).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Cyclin D1 antibody [SP4] - BSA and Azide free (AB239794)

Intracellular Flow Cytometry analysis of MCF-7 (human breast carcinoma) labeling Cyclin D1 with purified ab16663 at 1/30 dilution (9.03μg/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as a secondary antibody. Isotypecontrol - Rabbit monoclonal IgG (ab172730) (black). Unlableled control - Unlabelled cells (blue).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab16663).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Cyclin D1 antibody [SP4] - BSA and Azide free (AB239794)

Intracellular Flow Cytometry analysis of NIH/3T3 (mouse embryo) labeling Cyclin D1 with purified ab16663 at 1/30 dilution (9.03μg/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as a secondary antibody. Isotypecontrol - Rabbit monoclonal IgG (ab172730) (black). Unlabeled control - Unlabelled cells (blue).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab16663).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Cyclin D1 antibody [SP4] - BSA and Azide free (AB239794)

Immunocytochemistry/ Immunofluorescence analysis of C6 (rat glial tumor glial cell) cells labeling Cyclin D1 with purified ab16663 at 1/50 (5.42μg/ml). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 μg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 μg/ml) dilution. DAPI was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab16663).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cyclin D1 antibody [SP4] - BSA and Azide free (AB239794)

IHC image of ab16663 staining Cyclin D1 in rat esophagus formalin fixed paraffin embedded tissue sections, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab16663, 1 : 100 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the secondary only control (shown on the inset).

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab16663).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Cyclin D1 antibody [SP4] - BSA and Azide free (AB239794)

Immunocytochemistry/ Immunofluorescence analysis of Neuro-2a (mouse neuroblastoma neuroblast) cells labeling Cyclin D1 with purified ab16663 at 1/50 (5.42μg/ml). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 μg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 μg/ml) dilution. DAPI was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab16663).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Cyclin D1 antibody [SP4] - BSA and Azide free (AB239794)

Intracellular Flow Cytometry analysis of C6 (rat glioma) labeling Cyclin D1 with purified ab16663 at 1/30 dilution (9.03μg/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as a secondary antibody. Isotypecontrol - Rabbit monoclonal IgG (ab172730) (black). Unlableled control - Unlabelled cells (blue).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab16663).

• WB

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Western blot - Anti-Cyclin D1 antibody [SP4] - BSA and Azide free (AB239794)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab16663)

All lanes:

Western blot - Anti-Cyclin D1 antibody [SP4] (<a href='/en-us/products/primary-antibodies/cyclin-d1-antibody-sp4-ab16663'>ab16663</a>) at 1/1000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) cell lysate at 10 µg

Lane 2:

MCF7 (Human breast adenocarcinoma epithelial cell) cell lysate at 10 µg

Lane 3:

CCND1 KO HAP1 cell lysate at 10 µg

Lane 4:

HAP1 (Human chronic myelogenous leukemia near-haploid cell line) cell lysate at 10 µg

Lane 5:

Neuro-2a (Mouse neuroblastoma neuroblast) cell lysate at 10 µg

Lane 6:

NIH/3T3 (Mouse embryonic fibroblast) cell lysate at 10 µg

Lane 7:

C6 (Rat glial tumor glial cell) cell lysate at 10 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: 34 kDa

Observed band size: 33 kDa

false

Exposure time: 2s

• WB

Lab

Western blot - Anti-Cyclin D1 antibody [SP4] - BSA and Azide free (AB239794)

This data was developed using the same antibody clone in a different buffer formulation (ab16663).

Lanes 1- 2 : Merged signal (red and green). Green - ab16663 observed at 36 kDa. Red - Anti-Vinculin antibody [VIN-54] observed at 124 kDa.

ab16663 was shown to react with Cyclin D1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab255348 (knockout cell lysate ab263808) was used. Wild-type HeLa and CCND1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab16663 and Anti-Vinculin antibody [VIN-54] overnight at 4°C at a 1 in 200 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Cyclin D1 antibody [SP4] (<a href='/en-us/products/primary-antibodies/cyclin-d1-antibody-sp4-ab16663'>ab16663</a>) at 1/200 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

CCND1 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human CCND1 (Cyclin D1) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-ccnd1-cyclin-d1-knockout-hela-cell-line-ab255348'>ab255348</a>)

Predicted band size: 34 kDa

Observed band size: 36 kDa

false

• WB

Lab

Western blot - Anti-Cyclin D1 antibody [SP4] - BSA and Azide free (AB239794)

Lanes 1 - 3 : Merged signal (red and green). Green - ab16663 observed at 34 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab16663 was shown to specifically recognize CCND1 (Cyclin D1) in wild-type HAP1 cells as signal was lost at the expected MW in CCND1 (Cyclin D1) knockout cells. Wild-type and CCND1 (Cyclin D1) knockout samples were subjected to SDS-PAGE. ab16663 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 1/200 dilution and 1/20,000 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 1/20,000 dilution for 1 hour at room temperature before imaging.

This data was developed using the same antibody clone in a different buffer formulation containing Tris buffered saline, BSA, and sodium azide (ab16663).

All lanes:

Western blot - Anti-Cyclin D1 antibody [SP4] (<a href='/en-us/products/primary-antibodies/cyclin-d1-antibody-sp4-ab16663'>ab16663</a>)

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

CCND1 (Cyclin D1) knockout HAP1 whole cell lysate at 20 µg

Lane 3:

A431 whole cell lysate at 20 µg

Predicted band size: 34 kDa

false

• WB

Lab

Western blot - Anti-Cyclin D1 antibody [SP4] - BSA and Azide free (AB239794)

This data was developed using the same antibody clone in a different buffer formulation (ab16663).

Lanes 1- 2 : Merged signal (red and green). Green - ab16663 observed at 36 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

ab16663 was shown to react with Cyclin D1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab261760 (knockout cell lysate ab256864) was used. Wild-type HeLa and CCND1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab16663 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) overnight at 4°C at a 1 in 200 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Cyclin D1 antibody [SP4] (<a href='/en-us/products/primary-antibodies/cyclin-d1-antibody-sp4-ab16663'>ab16663</a>) at 1/200 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

CCND1 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human CCND1 (Cyclin D1) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-ccnd1-cyclin-d1-knockout-hela-cell-line-ab261760'>ab261760</a>)

Predicted band size: 34 kDa

Observed band size: 36 kDa

false

• WB

Supplier Data

Western blot - Anti-Cyclin D1 antibody [SP4] - BSA and Azide free (AB239794)

False colour image of Western blot : Anti-Cyclin D1 antibody [SP4] staining at 1/25 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab16663 was shown to bind specifically to Cyclin D1. A band was observed at 35 kDa in wild-type A549 cell lysates with no signal observed at this size in ccnd1 knockout cell line ab286759 (knockout cell lysate ab300213). To generate this image, wild-type and ccnd1 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution. This data was developed using the same antibody clone in a different buffer formulation (ab16663).

All lanes:

Western blot - Anti-Cyclin D1 antibody [SP4] (<a href='/en-us/products/primary-antibodies/cyclin-d1-antibody-sp4-ab16663'>ab16663</a>) at 1/25 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

Western blot - Human CCND1 knockout A549 cell lysate (ab300213) at 20 µg

Lane 3:

SH-SY5Y cell lysate at 20 µg

Lane 4:

K562 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 34 kDa

Observed band size: 35 kDa

false

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cyclin D1 antibody [SP4] - BSA and Azide free (AB239794)

Human mantle cell lymphoma stained with ab16663.

This image was generated from the hybridoma version.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab16663).