Flow cytometry overlay histogram showing wild-type Raji (green line) and CXCR5 knockout Raji cells (ab273380) stained with ab254415 (red line). The cells were incubated in 1x PBS containing 10μg/ml human IgG and 10% normal goat serumto block FC receptors and non-specific protein-protein interaction followed by the antibody (ab254415) (1x10⁶ in 100μl at 0.2 μg/ml) for 30 min at 4°C.

The secondary antibody Goat anti-rabbit IgG H&L (Alexa Fluor^® 488, pre-adsorbed) (ab150081) was used at 1/2000 for 30 min at 4°C.

Isotype control antibody was Rabbit IgG (monoclonal) (ab172730) used at the same concentration and conditions as the primary antibody (wild-type Raji - black line; CXCR5 knockout Raji - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling CXCR5 with ab254415 at 1/5000 dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human tonsil (PMID: 12393412). The section was incubated with ab254415 for 10 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

All lanes : Anti-CXCR5 antibody [EPR23463-30] (ab254415) at 1/1000 dilution

Lane 1 : Wild-type Raji cell lysate
Lane 2 : CXCR5 knockout Raji cell lysate
Lane 3 : Daudi cell lysate
Lane 4 : Jurkat cell lysate

Lysates/proteins at 30 µg per lane.

Performed under reducing conditions.

Predicted band size: 42 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab254415 observed at 60 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab254415 was shown to react with CXCR5 in Raji wild-type cells in Western blot with loss of signal observed in CXCR5 knockout sample. Wild-type and CXCR5 knockout Raji cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab254415 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 h at room temperature before imaging.

ab254415 staining CXCR5 in Daudi cells (top panel, positive control) and Jurkat cells (bottom panel, negative control). The cells were fixed with 4% paraformaldehyde (10 min) then 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 ab254415 at 5μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution 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) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

Flow cytometric analysis of Raji (Human Burkitt's lymphoma B lymphocyte) cells labelling CXCR5 with ab254415 at 1/500 dilution (0.1µg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody. Gated on viable cells.

Flow cytometric analysis of human peripheral blood mononuclear cell (PBMC) cells labelling CXCR5 with ab254415 at 1/500 dilution (0.1µg) (Right) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Left). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary at a 1/2000 dilution. Cells were stained with rabbit IgG (Left) or ab254415 (Right), then stained with anti-CD19 conjugated to Alexa Fluor^® 647.

Gated on viable cells.

All lanes : Anti-CXCR5 antibody [EPR23463-30] (ab254415) at 1/1000 dilution

Lane 1 : Neuro-2a (mouse neuroblastoma neuroblast), whole cell lysate
Lane 2 : A20 (mouse reticulum sarcoma B lymphocyte), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 42 kDa
Observed band size: 42 kDa

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

Samples are non-boiled as boiling may cause protein aggregates. 

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 30553016).

Exposure time: 20 seconds.

All lanes : Anti-CXCR5 antibody [EPR23463-30] (ab254415) at 1/1000 dilution

Lane 1 : Rat lymph node tissue lysate
Lane 2 : C6 (rat glial tumor glial cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 42 kDa
Observed band size: 42 kDa

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

This blot was developed using a higher sensitivity ECL substrate.

Samples are non-boiled as boiling may cause protein aggregates.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 30553016).

Exposure time: 122 seconds.

All lanes : Anti-CXCR5 antibody [EPR23463-30] (ab254415) at 1/1000 dilution

Lane 1 : Mouse spleen tissue lysate
Lane 2 : Mouse lymph node tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 42 kDa
Observed band size: 42 kDa

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

This blot was developed using a higher sensitivity ECL substrate.

Samples are non-boiled as boiling may cause protein aggregates.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 30553016).

Exposure time: 122 seconds.

Immunohistochemical analysis of paraffin-embedded human diffuse large B-cell lymphoma tissue labeling CXCR5 with ab254415 at 1/5000 dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human diffuse large B-cell lymphoma. The section was incubated with ab254415 for 10 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.