Flow Cytometry (Intracellular) - Anti-CD4 antibody [EPR6855] - BSA and Azide free (ab181724)

Human peripheral blood lymphocytes stained with unpurified ab133616 (red line). Human whole blood was processed using a modified protocol based on Chow et al, 2005 (PMID: 16080188). In brief, human whole blood was fixed in 4% formaldehyde (methanol-free) for 10 min at 22°C. Red blood cells were then lyzed by the addition of Triton X-100 (final concentration - 0.1%) for 15 min at 37°C. For experimentation, cells were treated with 50% methanol (-20°C) for 15 min at 4°C. Cells were then incubated with the antibody (unpurified ab133616, 1/100 dilution) for 30 min at 4°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 4°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of

30,000 total events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. Gating strategy - peripheral blood lymphocytes.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab133616).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD4 antibody [EPR6855] - BSA and Azide free (ab181724)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue labelling CD4 with purified ab133616 at 1/500. 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 (ab133616).

Western blot - Anti-CD4 antibody [EPR6855] - BSA and Azide free (ab181724)

This WB data was generated using the same anti-CD4 antibody clone [EPR6855] in a different buffer formulation (cat# ab133616).

Blocking buffer and concentration: 5% NFDM/TBST

Diluting buffer and concetration: 5% NFDM/TBST

All lanes:

Western blot - Anti-CD4 antibody [EPR6855] - BSA and Azide free (ab181724)

All lanes:

THP-1 (human monocytic leukemia cell line) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051)

Predicted band size: 51 kDa

Exposure time: 3min

Immunocytochemistry/ Immunofluorescence - Anti-CD4 antibody [EPR6855] - BSA and Azide free (ab181724)

This data was developed using the same antibody clone in a different buffer formulation (ab133616).

Immunocytochemistry analysis of THP-1 (Human monocytic leukemia monocyte) labeling CD4 with purified ab133616 at 1/100 dilution. Cells were fixed with 100% methanol. Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/1000 (2 µg/ml) was used as the secondary antibody. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 (2.30 µg/ml) was used as counterstain. Nuclei were stained blue with DAPI.

Negative control: PBS instead of the primary antibody.

Western blot - Anti-CD4 antibody [EPR6855] - BSA and Azide free (ab181724)

This data was developed using ab133616, the same antibody clone in a different buffer formulation. Different batches of ab133616 were tested on THP-1 (Human monocytic leukemia monocyte) lysate at 1.0 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 51 kDa.

All lanes:

Western blot - Anti-CD4 antibody [EPR6855] (ab133616)

Predicted band size: 51 kDa