Anti-PAX5 antibody [EPR3730(2)] (ab109443) at 1/5000 dilution (purified) + Mouse spleen tissue lysate at 20 µg

Secondary
Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

Predicted band size: 42 kDa
Observed band size: 45 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue labelling PAX5 with purified ab109443 at 1/1000. 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.

Immunocytochemistry/Immunofluorescence analysis of Ramos cells labelling PAX5 with purified ab109443 at 1/250. 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 (1/250) 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).

Chromatin was prepared from Ramos cells according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with 1.5 mM EGS for 30mins and then formaldehyde for 10min.
The ChIP was performed with 25 µg of chromatin, 5 µg of abzz (red), or 5 µg of rabbit normal IgG ab172730 (gray) and 25 µl of Protein A/G Dynabeads. The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
Primers and probes are from paper PMID:19806612
*http://www.abcam.com/resources?keywords=X%20ChIP%20protocol

ab109443 (purified) at 1/20 dilution immunoprecipitating PAX5 in mouse spleen lysate 10 µg.
Lane 1 (input): mouse spleen lysate 10 µg
Lane 2 (+): ab109443 & mouse spleen lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab109443 in mouse spleen lysate
For western blotting, ab109443 at 1/500 dilution (0.268 µg/mL) and veriBlot for IP secondary antibody (HRP) (ab131366) at 1/1000 dilution was used.

Blocking and diluting buffer: 5% NFDM /TBST.

ab109443 (purified) at 1/20 dilution immunoprecipitating PAX5 in Ramos (Human Burkitt's lymphoma B lymphocyte) whole cell lysate 10 µg.

Lane 1 (input): Ramos (Human Burkitt's lymphoma B lymphocyte) whole cell lysate 10 µg
Lane 2 (+): ab109443 & Ramos whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab109443 in Ramos whole cell lysate

For western blotting, ab109443 at 1/500 dilution (0.268 µg/mL) and veriBlot for IP secondary antibody (HRP) (ab131366) at 1/1000 dilution was used.

Blocking and diluting buffer: 5% NFDM /TBST.

Flow Cytometry analysis of Ramos cells labelling PAX5 with purified ab109443 at 1/30 (red). Cells were fixed with 80% methanol. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue labelling PAX5 with unpurified ab109443.

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

Anti-PAX5 antibody [EPR3730(2)] (ab109443) at 1/5000 dilution (purified) + Human tonsil tissue lysate at 20 µg

Secondary
Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/5000 dilution

Predicted band size: 42 kDa
Observed band size: 45 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Anti-PAX5 antibody [EPR3730(2)] (ab109443) at 1/20000 dilution (purified) + Ramos cell lysate at 20 µg

Secondary
Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

Predicted band size: 42 kDa
Observed band size: 45 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat spleen tissue labelling PAX5 with purified ab109443 at 1/1000. 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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human B-cell lymphoma tissue labelling PAX5 with purified ab109443 at 1/1000. 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.

All lanes : Anti-PAX5 antibody [EPR3730(2)] (ab109443) at 1/10000 dilution (unpurified)

Lane 1 : Ramos cell lysate
Lane 2 : Human tonsil tissue lysate
Lane 3 : Daudi cell lysates

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Standard HRP labelled goat anti-rabbit IgG at 1/2000 dilution

Predicted band size: 42 kDa

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human Hodgkin's lymphoma tissue labelling PAX5 with unpurified ab109443.

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

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human normal spleen tissue labelling PAX5 with unpurified ab109443.

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

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human diffuse B-cell lymphoma tissue labelling PAX5 with unpurified ab109443.

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

ab109443 showing negative staining in Normal human kidney tissue. 

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

Overlay histogram showing Ramos cells stained with unpurified ab109443 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween 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 ab109443, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

Equilibrium disassociation constant (K_D)
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