IHC image of Myelin Basic Protein staining in a section of formalin-fixed paraffin-embedded normal rat brain and normal rat pancreas, performed on a Leica BOND^TM. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6, epitope retrieval solution 1) for 20 minutes. The section was then incubated with ab209328, 1/1000 dilution, for 15 minutes at room temperature.

An HRP-conjugated goat anti-Human IgG secondary was used for 15 minutes at room temperature. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

ab209328 staining Myelin Basic Protein in SK-N-SH (Human neuroblastoma cell line) cells. The cells were fixed with 4% formaldehyde (10 minutes), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal donkey serum/0.3M glycine in 0.1% PBS-Tween for 1 hour. The cells were then incubated overnight at +4°C with ab209328 at a 5 µg/ml concentration, then detected with a donkey anti-human (Alexa Fluor^® 488) secondary antibody at a 1/2000 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue), and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 594), at a 1/250 dilution (shown in red).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab209328 staining Myelin Basic Protein in SHSY5Y cells. The cells were fixed with 100% methanol (5 minutes), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal donkey serum/0.3M glycine in 0.1% PBS-Tween for 1 hour. The cells were then incubated overnight at +4°C with ab209328 at a 5 µg/ml concentration, then detected with a donkey anti-human (Alexa Fluor^® 488) secondary antibody at a 1/1000 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue), and ab195884, Rat monoclonal to alpha Tubulin (Alexa Fluor^® 647), at a 1/250 dilution (shown in red).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

IHC image of Myelin Basic Protein staining in a section of formalin-fixed paraffin-embedded normal human hippocampus and normal human pancreas*, performed on a Leica BOND^TM. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6, epitope retrieval solution 1) for 20 minutes. The section was then incubated with ab209328, 1/1000 dilution, for 15 minutes at room temperature.

An HRP-conjugated goat anti-Human IgG secondary was used for 15 minutes at room temperature. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

 *Human pancreas tissue was obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

IHC image of Myelin Basic Protein staining in a section of formalin-fixed paraffin-embedded normal mouse brain and normal mouse pancreas, performed on a Leica BOND^TM. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6, epitope retrieval solution 1) for 20 minutes. The section was then incubated with ab209328, 1/1000 dilution, for 15 minutes at room temperature.

An HRP-conjugated goat anti-Human IgG secondary was used for 15 minutes at room temperature. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

All lanes : Anti-Myelin Basic Protein antibody [IGX3421] (ab209328) at 0.25 µg/ml

Lane 1 : Human brain tissue lysate - total protein (ab29466) at 10 µg
Lane 2 : Mouse brain tissue lysate at 10 µg
Lane 3 : Rat brain tissue lysate at 10 µg
Lane 4 : Myelin Basic Protein (Recombinant protein) at 0.1 µg

Secondary
All lanes : HRP conjugated Goat Anti-Human IgG (H+L) at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 33 kDa
Observed band size: 18,23,24 kDa why is the actual band size different from the predicted?

Exposure time :

Lane 1 : 30 seconds.

Lanes 2-3 : 2 minutes.

Lane 4 : 8 minutes.

This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% milk before being incubated with ab209328 overnight at 4°C. Antibody binding was visualised using ECL development solution ab133406.

ELISA using ab209328 for 16 hours at 4ºC. ab7153 goat anti human was used as a secondary at a 1/5000 dilution for 1 hour at Room Temperature.

Anti-Myelin Basic Protein antibody [IGX3421] (ab209328) at 1 µg/ml + Mouse brain tissue lysate at 10 µg

Secondary
HRP conjugated Goat Anti-Human IgG (H+L) at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 33 kDa
Observed band size: 20, 17 kDa why is the actual band size different from the predicted?


Exposure time: 2 minutes

This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% milk before being incubated with ab209328 overnight at 4°C. Antibody binding was visualised using ECL development solution ab133406.