Western blot - Myelin Basic Protein antibody (ab40390)

All lanes : Anti-Myelin Basic Protein antibody (ab40390) at 1 µg/ml

Lane 1 : Brain (Mouse) Tissue Lysate
Lane 2 : Brain (Rat) Tissue Lysate - normal tissue

Lysates/proteins at 10 µg per lane.

Secondary
Goat polyclonal to rabbit IgG - H&L - Pre adsorbed (HRP) at 1/3000 dilution

Performed under reducing conditions.

Predicted band size : 33 kDa
Observed band size : 18,23 kDa (why is the actual band size different from the predicted?)
Additional bands at : 45 kDa. We are unsure as to the identity of these extra bands.

This antibody was raised against an immunogen that is predicted to recognize isoforms (5, 7, 8, 10 and 13) of Myelin Basic Protein (MBP). The predicted molecular weights of isoforms (5, 7, 8, 10 and 13) are 18.5kDa, 17kDa, 14kDa, 21kDa and 13kDa respectively.

Immunocytochemistry/ Immunofluorescence - Myelin Basic Protein antibody (ab40390)

ICC/IF image of ab40390 stained PC12 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 (ab40390, 5µg/ml) overnight at +4ºC. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit 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).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Myelin Basic Protein antibody (ab40390)

ab40390 staining rat adult brain saggital tissue sections by IHC-P.  Sections were formaldehyde fixed and subjected to heat mediated antigen retrieval in citric acid (pH 6) prior to blocking in 1% BSA for 10 minutes at RT.  The primary antibody was diluted 1/100 and incubated with the sample for 16 hours.  A biotinylated goat anti-rabbit IgG antibody, diluted 1/300, was used as the secondary.

This image is courtesy of an Abreview submitted by Mr Carl Hobbs

Immunohistochemistry (Methylmethacrylate sections) - Myelin Basic Protein antibody (ab40390)

Immunohistochemical detection of Myelin Basic Protein antibody using (ab40390) on PFA perfusion fixed free-floating rat brain sections. Primary antibody used at 1/1000 and incubated for 18 hours @ 20°C in PBS + 0.3 % Triton X100. Secondary antibody: Goat anti-rabbit Alexa Fluor® 488 (1/1000).  Immunostaining is observed widely in tracts of axons, as expected. The pictures show the staining obtained at the level of the cerebral cortex, using the objective X5 (left) or X10 (right). The tissues were perfusion fixed with 4% PFA and later postfixed overnight in the same fixative. They were cryoprotected in 30% sucrose and cut using a cryostat.

Sophie Pezet, CNRS, Paris, France

Immunocytochemistry/ Immunofluorescence-Anti-Myelin Basic Protein antibody(ab40390)

ICC/IF image of ab40390 stained PC12 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 (ab40390, 5µg/ml) overnight at +4ºC. 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.