Anti-CD74 antibody

• IHC-P

AbReview18450****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD74 antibody (AB64772)

ab64772 (1/250) staining CD74 in paraffin-embedded Human tonsil tissue. Tissue underwent fixation in formaldehyde, peroxidase blocking, protein blocking and heat mediated antigen retrieval. The secondary antibody was goat anti rabbit conjugated to HRP. For further experimental details please refer to abreview.

This image is courtesy of an abreview submitted by Antibody Solutions Ltd.

• WB

Project5360****

Western blot - Anti-CD74 antibody (AB64772)

All lanes:

Western blot - Anti-CD74 antibody (ab64772) at 1 µg/mL

All lanes:

Raji (Human Burkitt's lymphoma cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Predicted band size: 34 kDa

Observed band size: 34 kDa

false

Exposure time: 2min

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD74 antibody (AB64772)

ab64772 (1 : 160) staining CD74 in paraffin-embedded human tonsil (left panel) using an automated system (Ventana Discovery). Right-hand panel shows negative control (no primary antibody).
Using this protocol there is strong membrane staining of activated B cells in the germinal centres and B cells of the mantle zone of the follicles plus scattered cells of the interfollicular areas (paracortical B cells).

Sections were rehydrated and antigen retrieved in CC1 Cell Conditioning Buffer using Ventana Mild Retrieval programme. Slides were blocked in 3% H₂O₂ / 4 min / 37°C and incubated with ab64772 (1 : 160 dilution / 2 hours / 37°C). Sections then blocked (4mins / 37°C) and incubated with Dako swine anti-rabbit antibody (1 : 50, 28 min / 37°C). Staining was amplified and detected by incubation with Ventana Streptavidin ABC system (16 min / 37°C) and Ventana DAB map reagent (8 min / 37°C). Slides were counterstained with Haematox

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-CD74 antibody (AB64772)

ICC/IF image of ab64772 stained RAW246.7 cells. The cells were 4% paraformaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab64772, 1µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD74 antibody (AB64772)

ab64772 (1 : 80) staining CD74 in paraffin-embedded human lymph node (left panel) using an automated system (Ventana Discovery). Right-hand panel shows negative control (no primary antibody).
Using this protocol there is strong membrane staining of activated B cells in the germinal centres and B cells of the mantle zone of the follicles plus scattered cells of the interfollicular areas (paracortical B cells).

Sections were rehydrated and antigen retrieved in CC1 Cell Conditioning Buffer using Ventana Mild Retrieval programme. Slides were blocked in 3% H₂O₂ / 4 min / 37°C and incubated with ab64772 (1 : 80 dilution / 1 hour / 37°C). Sections then blocked (3mins / 37°C) and incubated with Dako swine anti-rabbit antibody (1 : 50, 28 min / 37°C). Staining was amplified and detected by incubation with Ventana Streptavidin ABC system (16 min / 37°C) and Ventana DAB map reagent (8 min / 37°C). Slides were counterstained with Haematoxyli

• IP

Unknown

Immunoprecipitation - Anti-CD74 antibody (AB64772)

CD74 was immunoprecipitated using 0.5mg Raji whole cell extract, 5μg of Rabbit polyclonal to CD74 and 50μl of protein G magnetic beads (+). No antibody was added to the control (-).

The antibody was incubated under agitation with Protein G beads for 10min, Raji whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.

Proteins were eluted by addition of 40μl SDS loading buffer and incubated for 10min at 70°C; 10μl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab64772.

Secondary : Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).

Band : 34kDa; CD74

All lanes:

Immunoprecipitation - Anti-CD74 antibody (ab64772)

Predicted band size: 34 kDa

true

Exposure time: 12min

• WB

Lab

Western blot - Anti-CD74 antibody (AB64772)

Lanes 1 - 4 : Merged signal (red and green). Green - ab64772 observed at 35 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab64772 was shown to react with CD74 in western blot. The band observed in CD74 knockout cell line ab273378 (knockout lysate ab275529) below 35 kDa is likely to represent a truncated form. This has not been investigated further. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab64772 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at 1 μg/ml 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.

All lanes:

Western blot - Anti-CD74 antibody (ab64772) at 1 µg/mL

Lane 1:

Wild-type Raji cell lysate at 30 µg

Lane 2:

CD74 knockout Raji cell lysate at 30 µg

Lane 2:

Western blot - Human CD74 knockout Raji cell lysate (<a href='/en-us/products/cell-lysates/human-cd74-knockout-raji-cell-lysate-ab275529'>ab275529</a>)

Lane 2:

Western blot - Human CD74 knockout Raji cell line (<a href='/en-us/products/cell-lines/human-cd74-knockout-raji-cell-line-ab273378'>ab273378</a>)

Lane 3:

Jurkat cell lysate at 30 µg

Lane 4:

HepG2 cell lysate at 30 µg

Predicted band size: 34 kDa

Observed band size: 35 kDa

false

• WB

Lab

Western blot - Anti-CD74 antibody (AB64772)

Lanes 1 - 4 : Merged signal (red and green). Green - ab64772 observed at 35 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab64772 was shown to react with CD74 in wild-type Raji cells in Western blot with loss of signal observed in CD74 knockout cell line ab273876 (knockout cell lysate ab273830). Wild-type Raji and CD74 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab64772 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at 1 μg/ml 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.

All lanes:

Western blot - Anti-CD74 antibody (ab64772) at 1 µg/mL

Lane 1:

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

Lane 2:

CD74 knockout Raji (Human Burkitt's lymphoma cell line) whole cell lysate at 20 µg

Lane 2:

Western blot - Human CD74 knockout Raji cell line (<a href='/en-us/products/cell-lines/human-cd74-knockout-raji-cell-line-ab273876'>ab273876</a>)

Lane 3:

Daudi (Human Burkitt's lymphoma cell line) whole cell lysate at 20 µg

Lane 4:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg

Predicted band size: 34 kDa

Observed band size: 35 kDa

false