Anti-Glucose Transporter GLUT4 antibody (ab33780) at 1 µg/ml + Partial tagged recombinant protein to GLUT4 at 0.1 µg

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

Predicted band size: 55 kDa
Observed band size: 30 kDa why is the actual band size different from the predicted?

ab33780 gave a positive signal against the partial recombinant GLUT4 protein which has an expected molecular weight of 30 kDa.

Immunofluorescence staining of GLUT4 using ab33780 in ioSkeletal Myocytes - Human iPSC-Derived Skeletal Myocytes (ab277612), which were differentiated for 3 (left panel), 5 (middle panel) and 10 days (right panel) post induction.

The cells were fixed with 100% MeOH (5 min) and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab33780 at 5 µg/mL and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin, at 1/1000 dilution. Cells were then incubated with ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150120, Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown. Gamma is adjusted to 1.5 in all channels.

ICC/IF image of ab33780 stained HeLa 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 (ab33780, 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 image of Glucose Transporter GLUT4 staining in Human normal heart muscle formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab33780, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

 

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.

Immunohistochemical of PFA-fixed paraffin-embedded rat skeletal muscle skeletal tissue, labelling glucose transporter GLUT4 with ab33780 at a dilution of 1/200 incubated for 13 hours at 4°C in 1% BSA in TBS. Antigen retrival was with Tris-EDTA at pH 9.0 (heat mediated). Blocking was with 3% BSA incubated for 1 hour at 37°C. Secondary was a Goat anti-rabbit polyclonal Akaline Phosphotase conjugate at 1/200.

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All lanes : Anti-Glucose Transporter GLUT4 antibody (ab33780) at 1 µg/ml

Lane 1 : Recombinant Protein GLUT4 (Partial, Tagged) at 0.1 µg
Lane 2 : Heart (Human) Tissue Lysate at 20 µg
Lane 3 : Skeletal Muscle (Human) Tissue Lysate at 20 µg

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: 55 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?


Exposure time: 4 minutes

IHC image of Glucose Transporter GLUT4 staining in Mouse normal skeletal muscle formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol B. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab33780, 5µg/ml, for 15 mins at room temperature. A Goat anti-Rabbit biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

 

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.

ab33780 staining glucose transporter GLUT4 in human heart tissue section by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue underwent fixation in formaldehyde, heat mediated antigen retrieval in Citrate buffer pH 6.0 and blocking (5 minutes/peroxidase block then 10 minutes/protein block) for 15 minutes at 20°C. The primary antibody was diluted, 1/2000 and incubated with sample for 45 minutes at 20°C. A HRP conjugated goat polyclonal to rabbit IgG was used undiluted as secondary.

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