Anti-CD86 antibody [EP1158-37]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD86 antibody [EP1158-37] (AB269587)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling CD86 with ab269587 at 1/100 dilution (2.52 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human tonsil (PMID : 22740445). The section was incubated with ab269587 for 30 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 a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD86 antibody [EP1158-37] (AB269587)

Immunohistochemical analysis of paraffin-embedded Human non-Hodgkin lymphoma tissue labeling CD86 with ab269587 at 1/100 dilution (2.52 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human non-Hodgkin lymphoma (PMID : 9767448,10089910). The section was incubated with ab269587 for 30 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 a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

• WB

Lab

Western blot - Anti-CD86 antibody [EP1158-37] (AB269587)

Lanes 1 - 2 : Merged signal (red and green). Green - ab269587 observed at 70 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.

ab269587 was shown to react with CD86 in wild-type Raji cells in western blot with loss of signal observed in CD86 knockout sample. Wild-type and CD86 knockout Raji cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab269587 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 hour at room temperature before imaging.

All lanes:

Western blot - Anti-CD86 antibody [EP1158-37] (ab269587) at 1/1000 dilution

Lane 1:

Wild-type Raji (Human Burkitt's lymphoma cell line) whole cell lysate at 20 µg

Lane 2:

CD86 knockout Raji (Human Burkitt's lymphoma cell line) whole cell lysate at 20 µg

Lane 2:

Western blot - Human CD86 knockout Raji cell line (<a href='/en-us/products/cell-lines/human-cd86-knockout-raji-cell-line-ab273858'>ab273858</a>)

Predicted band size: 38 kDa

Observed band size: 70 kDa

false

• WB

Unknown

Western blot - Anti-CD86 antibody [EP1158-37] (AB269587)

Blocking and dilution buffer : 5% NFDM/TBSt.

Exposure time : 3 minutes.

Negative control : Jurkat (PMID : 9973476).

The actual band size higher than predicted is mainly due to glycosylation.

The molecular weight observed is consistent with what has been described in the literature (PMID : 27464357, 21896490).

All lanes:

Western blot - Anti-CD86 antibody [EP1158-37] (ab269587) at 1/1000 dilution

Lane 1:

Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate at 20 µg

Lane 2:

Daudi (human burkitt's lymphoma lymphoblast) whole cell lysate at 20 µg

Lane 3:

Jurkat (human t cell leukemia t lymphocyte) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 38 kDa

Observed band size: 80 kDa

false

• WB

Unknown

Western blot - Anti-CD86 antibody [EP1158-37] (AB269587)

Blocking and dilution buffer : 5% NFDM/TBST.

Exposure time : 3 minutes.

The actual band size higher than predicted is mainly due to glycosylation.

The molecular weight observed is consistent with what has been described in the literature (PMID : 27464357, 21896490).

This blot was developed using a higher sensitivity ECL substrate.

All lanes:

Western blot - Anti-CD86 antibody [EP1158-37] (ab269587) at 1/1000 dilution

Lane 1:

Human tonsil lysate at 20 µg

Lane 2:

Human spleen lysate at 20 µg

Secondary

All lanes:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Predicted band size: 38 kDa

Observed band size: 80 kDa

false