Immunofluorescent analysis of Spectrin beta-1 (green) showing staining in the cytoplasm of Hela cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a Spectrin beta-1 monoclonal antibody (Product # MA3-062) in 3% BSA-PBS at a dilution of 1:100 and incubated overnight at 4 °C in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a flourescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.
|Tested species reactivity||Human, Mouse, Rat|
|Published species reactivity||Rat|
|Host / Isotype||Mouse / IgG1|
|Immunogen||Purified human erythrocyte beta-1 spectrin.|
|Storage buffer||ascites diluted in PBS|
|Contains||0.05% sodium azide|
|Storage Conditions||-20° C, Avoid Freeze/Thaw Cycles|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||0.5-2 ug/test|
|Immunohistochemistry (Paraffin) (IHC (P))||1:20-1:200|
|Western Blot (WB)||1:100|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
|Immunohistochemistry (IHC)||See 1 publications below|
MA3-062 detects spectrin from mouse, rat and human erythrocytes, brain and muscle cells. This antibody has been shown to specifically detect the two known alternatively spliced forms of spectrin, beta-1 epsilon-1, present in erythrocytes, and beta-1 epsilon-2, present in nerve and striated muscle cells. It does not cross-react with alpha-2 spectrin or either of the fodrin subunits.
MA3-062 has been successfully used in Western blot, immunohistochemistry (paraffin) and immunofluorescence procedures. By Western blot, this antibody detects a ~246 kDa protein representing beta-1 spectrin in rat skeletal muscle homogenate. Immunofluorescence staining of beta-1 spectrin in rat skeletal muscle fibers with MA3-062 results in intense staining of the sarcolemma.
In immunofluorescence, antigen integrity can be compromised if aldehyde fixatives are left in contact with the protein for extended periods of time. If paraformaldehyde is used as a fixative, exposure should be limited to 5 minutes or less of no more than a 2% solution.
The MA3-062 antigen is purified human erythrocyte beta-1 spectrin.
Spectrin (Sp), the most abundant of the erythrocyte membrane skeleton proteins, helps these cells maintain their characteristic biconcave shape while remaining flexible and elastic. Erythrocyte Sp is a heterodimer composed of a 280 kDa alpha subunit and a 246 kDa beta subunit which associate in a side-to-side, antiparallel configuration to form a 100 nm rod-like structure. Sp in other tissues may be composed of distinct but homologous alpha and beta subunits, sometimes referred to as fodrin.
A newly introduced nomenclature designates the Sp subunits of the erythrocyte as alpha-1 and beta-1, and the fodrin subunits as alpha-2 and beta-2. Alternatively spliced forms of each are designated as epsilon-1, epsilon-2, etc. (e.g. beta-1 epsilon-1).
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Two populations of beta-spectrin in rat skeletal muscle.
MA3-062 was used in immunohistochemistry to evaluate the interaction between alpha fodrin and muscle beta spectrin
|Porter GA,Scher MG,Resneck WG,Porter NC,Fowler VM,Bloch RJ||Cell motility and the cytoskeleton (37:7)||1997|