Anti-CD58 antibody [EPR24012-147]

• Flow Cyt

Unknown

Flow Cytometry - Anti-CD58 antibody [EPR24012-147] (AB275392)

Flow cytometric analysis of Human peripheral blood mononuclear (PBMC) cells labelling CD58 with ab275392 at 1/500 dilution (0.1ug) (Right) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Left).

Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Gated on viable cells.

• Flow Cyt

Lab

Flow Cytometry - Anti-CD58 antibody [EPR24012-147] (AB275392)

Flow cytometry staining of human peripheral blood mononuclear cells (PBMCs) with ab275392 (right) or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (left). PBMCs were incubated for 30 min on ice in 1x PBS containing 10 µg/ml human IgG and 10 % normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody ab275392 or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (1x 10⁶ in 100 µl at 1 μg/ml (1/2,300)) for 30min on ice. The cells were simultaneously stained with CD56.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed was incubated at 1/4000 for 30min on ice

Acquisition of >30000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. Events were gated on live cells.

• WB

Lab

Western blot - Anti-CD58 antibody [EPR24012-147] (AB275392)

Western blot : Anti-CD58 antibody [EPR24012-147] ab275392 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 55-70 kDa in Wild-type Raji cell lysates with no signal observed at this size in CD58 knockout Raji cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-CD58 antibody [EPR24012-147] (ab275392) at 1/1000 dilution

Lane 1:

Wild-type Raji at 20 µg

Lane 2:

Western blot - Human CD58 knockout Raji cell line (<a href='/en-us/products/cell-lines/human-cd58-knockout-raji-cell-line-ab290422'>ab290422</a>) at 20 µg

Lane 3:

Wild-type HeLa at 20 µg

Lane 4:

CD58 knockout HeLa cells at 20 µg

Secondary

Lanes 1 - 4:

Goat anti-Rabbit 800CW at 1/20000 dilution

Lanes 1 - 4:

Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 28 kDa,52-60 kDa

Observed band size: 55-70 kDa,37 kDa

false

• Flow Cyt

Unknown

Flow Cytometry - Anti-CD58 antibody [EPR24012-147] (AB275392)

Flow cytometric analysis of A-204 (Human muscle rhabdomyosarcoma, Left) / Jurkat (Human T cell leukemia T lymphocyte, Right) cells labelling CD58 with ab275392 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Black) 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.

Low expression control : A-204.

Gated on viable cells.

• IP

Unknown

Immunoprecipitation - Anti-CD58 antibody [EPR24012-147] (AB275392)

CD58 was immunoprecipitated from 0.35 mg Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate with ab275392 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab275392 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate 10 ug

Lane 2 : 275392 IP in Raji whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab275392 in Raji whole cell lysate

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

Exposure time : 15 seconds.

This blot was developed using a higher sensitivity ECL substrate.

All lanes:

Immunoprecipitation - Anti-CD58 antibody [EPR24012-147] (ab275392)

Predicted band size: 28 kDa

Observed band size: 55-70 kDa

false

• WB

Lab

Western blot - Anti-CD58 antibody [EPR24012-147] (AB275392)

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

The molecular weight observed is consistent with what has been described in the literature (PMID : 26039213, 3309127, 19268704).

Low expression : A-204 (PMID : 26039213).

Exposure times : Lane 1 : 37 seconds; Lanes 2-3 : 15 seconds; Lanes 4-5 : 70 seconds.

All lanes:

Western blot - Anti-CD58 antibody [EPR24012-147] (ab275392) at 1/1000 dilution

Lane 1:

Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate at 40 µg

Lane 2:

A-204 (human muscle rhabdomyosarcoma) whole cell lysate at 40 µg

Lane 3:

K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 40 µg

Lane 4:

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

Lane 5:

TT (human thyroid carcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 28 kDa

Observed band size: 55-70 kDa

false

• WB

Lab

Western blot - Anti-CD58 antibody [EPR24012-147] (AB275392)

False colour image of Western blot : Anti-CD58 antibody [EPR24012-147] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab275392 was shown to bind specifically to CD58. A band was observed at 55 kDa in wild-type HeLa cell lysates with no signal observed at this size in CD58 knockout cell line ab265947 (knockout cell lysate ab257880). To generate this image, wild-type and CD58 knockout HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged.Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-CD58 antibody [EPR24012-147] (ab275392) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

CD58 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human CD58 knockout HeLa cell lysate (<a href='/en-us/products/cell-lysates/human-cd58-knockout-hela-cell-lysate-ab257880'>ab257880</a>)

Lane 2:

Western blot - Human CD58 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-cd58-knockout-hela-cell-line-ab265947'>ab265947</a>)

Lane 3:

Raji cell lysate at 20 µg

Lane 4:

SH-SY5Y cell lysate at 20 µg

Predicted band size: 28 kDa

Observed band size: 55 kDa

false

• WB

Lab

Western blot - Anti-CD58 antibody [EPR24012-147] (AB275392)

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

The molecular weight observed is consistent with what has been described in the literature (PMID : 26039213, 3309127, 19268704).

Exposure time : 3 minutes.

All lanes:

Western blot - Anti-CD58 antibody [EPR24012-147] (ab275392) at 1/1000 dilution

Lane 1:

Human kidney tissue lysate at 20 µg

Lane 2:

Human placenta tissue lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 28 kDa

Observed band size: 55-70 kDa

false