ICC/IF image of ab133133 stained MCF7 cells. The cells were 100% methanol fixed (5 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 (ab133133, 10µg/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. This antibody also gave a positive result in 4% formaldehyde fixed (10 min) Hek293 and MCF7 cells at 10µg/ml.

All lanes : Anti-PHGDH/Malate dehydrogenase antibody (ab133133) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
Lane 3 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 4 : Raji (Human Burkitt's lymphoma cell line) Whole Cell Lysate
Lane 5 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 56 kDa


Exposure time: 5 minutes


This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 5% Bovine Serum Albumin before being incubated with ab133133 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.