Western blot - ADAMTS5 antibody (ab41037)

All lanes : Anti-ADAMTS5 antibody (ab41037) at 1/250 dilution

Lane 1 : Placenta (Human) Tissue Lysate - adult normal tissue (ab29745)
Lane 2 : Placenta (Mouse) Whole Cell Lysate - normal tissue (ab29139)
Lane 3 : Uterus (Human) Whole Cell Lysate - normal tissue
Lane 4 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size : 102 kDa
Observed band size : 75 kDa (why is the actual band size different from the predicted?)
Additional bands at : 27 kDa. We are unsure as to the identity of these extra bands.

ab41037 appears to detect at 73kDa cleaved form of ADAMTS5. The precursor of this secreted protein is cleaved at a furin cleavage site by a furin endopeptidase. The doublet observed in human uterus lysate is possibly due to glycosylation.

Western blot - ADAMTS5 antibody (ab41037)

All lanes : Anti-ADAMTS5 antibody (ab41037) at 1/250 dilution

Lane 1 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate (ab7179)
Lane 2 : MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
Lane 3 : Brain (Mouse) Tissue Lysate
Lane 4 : Liver (Mouse) Tissue Lysate - normal tissue
Lane 5 : Testis (Mouse) Tissue Lysate - normal tissue
Lane 6 : Spinal Cord (Mouse) Tissue Lysate
Lane 7 : Ovary (Mouse) Tissue Lysate - normal tissue
Lane 8 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate
Lane 9 : Brain (Rat) Tissue Lysate - normal tissue
Lane 10 : Liver (Rat) Tissue Lysate

Lysates/proteins at 10 µg per lane.

Secondary
IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size : 102 kDa
Observed band size : 74 kDa (why is the actual band size different from the predicted?)


ab41037 appears to detect a 74 kDa cleaved form of ADAMTS5. The precursor of this secreted protein is cleaved at a furin cleavage site by a furin endopeptidase.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - ADAMTS5 antibody (ab41037)

ab41037 staining ADAMTS5 in mouse knee joint tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue was fixed with formaldehyde and an enzymatic mediated antigen retrieval step was performed using Proteinase K. Samples were then blocked with 5% serum for 10 minutes at room temperature followed by incubation with the primary antibody at 10µg/ml for 1 hour. An undiluted HRP-conjugated human anti-rabbit monoclonal was used as secondary antibody.

This image is a courtesy of an anonymous Abreview

Immunocytochemistry/ Immunofluorescence - Anti-ADAMTS5 antibody (ab41037)

ICC/IF image of ab41037 stained MEF1 cells. The cells were 4% formaldehyde 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 ab41037 at 5µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/1000 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.