重组Anti-CD58抗体[EPR27435-85]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CD58 antibody [EPR27435-85] (AB308459)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized CD58 KO HeLa (CD58 knockout human cervix adenocarcinoma epithelial cell)(ab265947); Parental HeLa (parental human cervix adenocarcinoma epithelial cell) cells labelling CD58 with ab308459 at 1/50 (10.08 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green). Confocal image showing membranous staining in parental HeLa, and no staining CD58 KO HeLa cell line. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-CD58 antibody [EPR27435-85] (AB308459)

Flow cytometric analysis of CD58 KO HeLa (ab265947) (human cervical adenocarcinoma epithelial cell, Left) / Parental HeLa (Right) cells labelling CD58 with ab308459 at 1/500 dilution (0.1 ug)/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, ab150081) at 1/5000 dilution was used as the secondary antibody. Gated on viable cells.

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-CD58 antibody [EPR27435-85] (AB308459)

Flow cytometric analysis of Human PBMC (human peripheral blood mononuclear cell) cells labelling CD58 with ab308459 at 1/500 dilution (0.1 ug)/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, ab150081) at 1/5000 dilution was used as the secondary antibody. Cells were co-stained with anti-CD3 conjugated to 647. Gated on viable cells.

• IP

Supplier Data

Immunoprecipitation - Anti-CD58 antibody [EPR27435-85] (AB308459)

CD58 was immunoprecipitated from 0.35 mg Raji (human burkitt's lymphoma b lymphocyte) whole cell lysate 10 ug with ab308459 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab308459 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Raji whole cell lysate

Lane 2 : ab308459 IP in Raji whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab308459 in Raji whole cell lysate

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

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

All lanes:

Immunoprecipitation - Anti-CD58 antibody [EPR27435-85] (ab308459) at 1/30 dilution

All lanes:

Raji (human burkitt's lymphoma b lymphocyte) whole cell lysate at 10 µg

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 55 kDa,70 kDa

true

Exposure time: 102s

• WB

Supplier Data

Western blot - Anti-CD58 antibody [EPR27435-85] (AB308459)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. The molecular weight observed is consistent with what has been described in the literature (PMID : 19268704; PMID : 3309127) and due to the high glycosylation of the protein. The blot of lane 1 was developed using a high sensitivity ECL substrate. Exposure time : 180 seconds.

All lanes:

Western blot - Anti-CD58 antibody [EPR27435-85] (ab308459) at 1/1000 dilution

Lane 1:

Human kidney tissue lysate at 20 µg

Lane 2:

Human tonsil tissue lysate at 20 µg

Lane 3:

Human placenta tissue lysate at 20 µg

Lane 4:

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

Lane 5:

K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

Secondary

Lanes 1 - 5:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/2000 dilution

Lanes 1 - 5:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/20000 dilution

Predicted band size: 28 kDa

Observed band size: 55-70 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-CD58 antibody [EPR27435-85] (AB308459)

Blocking and diluting buffer and concentration : Intercept® (TBS) Blocking Buffer diluted with an equal volume of TBS. Lysates at 20 µg per lane. The samples were run on a Bis-Tris gel under reducing conditions. Western blot : Anti-CD58 antibody (ab308459) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20, 000 dilution, shown in red. In Western blot, ab308459 was shown to bind specifically to CD58. Target of interest was observed at 55-70 kDa in wild-type HeLa cell lysates (lane 1) with no signal observed at this size in CD58 knockout cell line ab265947 (knockout cell lysate ab257880)(lane 2). To generate this image, samples were first run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in a fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4 °C. Blots were washed in TBS-T, incubated with secondary antibodies Goat anti-Rabbit IgG H&L 800CW (ab216773) and Goat anti-Mouse IgG H&L 680RD (ab216776) at 1/20000 dilution for 1 h at room temperature, washed again then imaged. Low expression : A-204 (PMID : 26039213).

All lanes:

Western blot - Anti-CD58 antibody [EPR27435-85] (ab308459) at 1/1000 dilution

Lane 1:

Wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

CD58 knockout HeLa whole cell lysate at 20 µg

Lane 3:

Raji (human burkitt's lymphoma b lymphocyte) whole cell lysate at 20 µg

Lane 4:

A-204 (human muscle rhabdomyosarcoma cell) whole cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Lanes 1 - 4:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution

Predicted band size: 28 kDa

Observed band size: 55-70 kDa

false