Anti-PAX5 抗体 [EPR3730(2)]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAX5 antibody [EPR3730(2)] (AB109443)

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.

• WB

Lab

Western blot - Anti-PAX5 antibody [EPR3730(2)] (AB109443)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-PAX5 antibody [EPR3730(2)] (ab109443) at 1/5000 dilution

All lanes:

Human tonsil tissue lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 29 kDa,42 kDa

Observed band size: 28 kDa,45 kDa

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• WB

Lab

Western blot - Anti-PAX5 antibody [EPR3730(2)] (AB109443)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-PAX5 antibody [EPR3730(2)] (ab109443) at 1/5000 dilution

All lanes:

Mouse spleen tissue lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 42 kDa

Observed band size: 45 kDa

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• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-PAX5 antibody [EPR3730(2)] (AB109443)

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 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-PAX5 antibody [EPR3730(2)] (AB109443)

Intracellular 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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAX5 antibody [EPR3730(2)] (AB109443)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAX5 antibody [EPR3730(2)] (AB109443)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAX5 antibody [EPR3730(2)] (AB109443)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAX5 antibody [EPR3730(2)] (AB109443)

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-PAX5 antibody [EPR3730(2)] (AB109443)

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).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAX5 antibody [EPR3730(2)] (AB109443)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAX5 antibody [EPR3730(2)] (AB109443)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAX5 antibody [EPR3730(2)] (AB109443)

ab109443 showing negative staining in Normal human kidney tissue.

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

• IP

Lab

Immunoprecipitation - Anti-PAX5 antibody [EPR3730(2)] (AB109443)

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.

All lanes:

Immunoprecipitation - Anti-PAX5 antibody [EPR3730(2)] (ab109443)

Predicted band size: 42 kDa

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• ChIP

Lab

ChIP - Anti-PAX5 antibody [EPR3730(2)] (AB109443)

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

• IP

Lab

Immunoprecipitation - Anti-PAX5 antibody [EPR3730(2)] (AB109443)

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.

All lanes:

Immunoprecipitation - Anti-PAX5 antibody [EPR3730(2)] (ab109443)

Predicted band size: 42 kDa

false

• WB

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Western blot - Anti-PAX5 antibody [EPR3730(2)] (AB109443)

All lanes:

Western blot - Anti-PAX5 antibody [EPR3730(2)] (ab109443) at 1/10000 dilution

Lane 1:

Ramos cell lysate at 10 µg

Lane 2:

Human tonsil tissue lysate at 10 µg

Lane 3:

Daudi cell lysates at 10 µg

Secondary

All lanes:

Standard HRP labelled goat anti-rabbit IgG at 1/2000 dilution

Predicted band size: 42 kDa

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• WB

Lab

Western blot - Anti-PAX5 antibody [EPR3730(2)] (AB109443)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-PAX5 antibody [EPR3730(2)] (ab109443) at 1/20000 dilution

All lanes:

Ramos cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 42 kDa

Observed band size: 45 kDa

false

• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-PAX5 antibody [EPR3730(2)] (AB109443)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.