Anti-p21 antibody [EPR362] - BSA and Azide free

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p21 antibody [EPR362] - BSA and Azide free (AB218311)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human papillary carcinoma of the thyroid gland tissue labelling p21 with unpurified ab109520 at a dilution of 1/100.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109520).

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-p21 antibody [EPR362] - BSA and Azide free (AB218311)

Cell line MCF7 (Human breast adenocarcinoma cell line), Target AbID ab109520 anti-p21 ab150077 Alexa Fluor^® 488 Goat anti-Rabbit secondary. Counterstain AbID ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594). Fixative 4% Paraformaldehyde, Permeabilisation 0.1% tritonX-100, Nuclear counter stain DAPI. Comments Confocal image showing nuclear staining on MCF7 cell line. Target 1oAb dilution 1 : 500 2 μg/ml, Target 2ndry Ab dilution 1 : 1000 2 μg/ml, Counterstain Ab dilution 1 : 200 2.5 μg/ml.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109520).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-p21 antibody [EPR362] - BSA and Azide free (AB218311)

Immunocytochemistry/Immunofluorescence analysis of MCF7 cells labelling p21 with purified ab109520 at 1/1000. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500) were also used.

Control 1 : primary antibody (1/1000) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Control 2 : ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109520).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p21 antibody [EPR362] - BSA and Azide free (AB218311)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human thyroid carcinoma tissue labelling p21 with purified ab109520 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109520).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p21 antibody [EPR362] - BSA and Azide free (AB218311)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervical carcinoma tissue labelling p21 with unpurified ab109520 at a dilution of 1/100.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109520).

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-p21 antibody [EPR362] - BSA and Azide free (AB218311)

Overlay histogram showing HeLa cells stained with unpurified ab109520 (red line). The cells were fixed with 80% methanol (5 min) then permeabilized with 0.1% PBS-Tween 20 for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (unpurified ab109520, 1/100) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150081) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (ab172730, 1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109520).

• IP

Lab

Immunoprecipitation - Anti-p21 antibody [EPR362] - BSA and Azide free (AB218311)

ab109520 (purified) at 1/50 immunoprecipitating p21 in HEK293 whole cell lysate.

Lane 1 (input) : HEK293 whole cell lysate (10μg)

Lane 2 (+) : ab109520 + HEK293 whole cell lysate (10μg).

Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab109520 in HEK293 whole cell lysate.

For western blotting, ab131366 VeriBlot for IP (HRP) was used for detection at 1/1500 dilution.

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109520).

All lanes:

Immunoprecipitation - Anti-p21 antibody [EPR362] (<a href='/en-us/products/primary-antibodies/p21-antibody-epr362-ab109520'>ab109520</a>)

Predicted band size: 18 kDa

Observed band size: 21 kDa

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• WB

Lab

Western blot - Anti-p21 antibody [EPR362] - BSA and Azide free (AB218311)

False colour image of Western blot : Anti-p21 antibody [EPR362] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab109520 was shown to bind specifically to p21. A band was observed at 21 kDa in wild-type HeLa cell lysates with no signal observed at this size in CDKN1A knockout cell line ab255349 (knockout cell lysate ab263812). To generate this image, wild-type and CDKN1A knockout y 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 (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109520).

All lanes:

Western blot - Anti-p21 antibody [EPR362] (<a href='/en-us/products/primary-antibodies/p21-antibody-epr362-ab109520'>ab109520</a>) at 1/1000 dilution

Lane 1:

wild-type HeLa Vehicle Control Fluvastatin (20 uM, 24 h) cell lysate at 20 µg

Lane 2:

wild-type HeLa Treated Fluvastatin (50 uM, 24 h) cell lysate at 20 µg

Lane 3:

CDKN1A knockout HeLa Vehicle Control Fluvastatin (20 uM, 24 h) cell lysate at 20 µg

Lanes 3 - 4:

Western blot - Human CDKN1A knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-cdkn1a-knockout-hela-cell-line-ab255349'>ab255349</a>)

Lane 4:

CDKN1A knockout HeLa Treated Fluvastatin (50 uM, 24 h) cell lysate at 20 µg

Lane 5:

MCF7 cell lysate at 20 µg

Lane 6:

SH-SY5Y cell lysate at 20 µg

Predicted band size: 18 kDa

Observed band size: 21 kDa

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• WB

Lab

Western blot - Anti-p21 antibody [EPR362] - BSA and Azide free (AB218311)

This data was developed using ab109520, the same antibody clone in a different buffer formulation. Different batches of ab109520 were tested on MCF7 (Human breast adenocarcinoma epithelial cell) lysate at 0.2 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 21 kDa.

All lanes:

Western blot - Anti-p21 antibody [EPR362] (<a href='/en-us/products/primary-antibodies/p21-antibody-epr362-ab109520'>ab109520</a>)

Predicted band size: 18 kDa

false

• WB

Lab

Western blot - Anti-p21 antibody [EPR362] - BSA and Azide free (AB218311)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109520). Western blot : Anti-CDKN1A antibody [EPR362] (ab109520) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab109520 was shown to bind specifically to CDKN1A. A band was observed at 21 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in CDKN1A knockout cell line. To generate this image, wild-type and CDKN1A knockout HCT 116 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.

All lanes:

Western blot - Anti-p21 antibody [EPR362] (<a href='/en-us/products/primary-antibodies/p21-antibody-epr362-ab109520'>ab109520</a>) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

Western blot - Human CDKN1A knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-cdkn1a-knockout-hct116-cell-line-ab288187'>ab288187</a>)

Lane 2:

CDKN1A knockout HCT 116 cell lysate at 20 µg

Lane 3:

Wild-type MCF7 cell lysate at 20 µg

Lane 4:

Western blot - Human CDKN1A knockout MCF7 cell line (<a href='/en-us/products/cell-lines/human-cdkn1a-knockout-mcf7-cell-line-ab288200'>ab288200</a>)

Lane 4:

CDKN1A knockout MCF7 cell lysate at 20 µg

Lane 5:

Wild-type A549 cell lysate at 20 µg

Lane 6:

Western blot - Human CDKN1A knockout A549 cell line (<a href='/en-us/products/cell-lines/human-cdkn1a-knockout-a549-cell-line-ab288213'>ab288213</a>)

Lane 6:

CDKN1A knockout A549 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: 21 kDa

Observed band size: 21 kDa

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