Enriched rabbit α-DG (100 μl of the 150 mM sodium acetate (pH 5.5) solution) was mixed with Bgus (0.45 U) and/or Xylsa (0.09 U), or no enzymes, and incubated overnight at 65°C. Samples were then run on SDS-PAGE, transferred to PVDF-FL (Millipore), and probed with anti-α-DG core antibody (AF6868) and anti-α-DG glycan antibody (IIH6). Enriched rabbit α-DG (100 μl of the 150 mM sodium acetate (pH 5.5) solution) was mixed with Bgus (0.45 U) and/or Xylsa (0.09 U), or no enzymes, and incubated overnight at 65°C. Samples were then run on SDS-PAGE, transferred to PVDF-FL (Millipore), and probed with anti-α-DG core antibody (AF6868), rabbit polyclonal antibody against the α2 subunit of the DHPR Ca2+ channel41 (link) (Campbell lab) and anti-α-DG glycan antibodies (IIH6, VIA41). The laminin overlay assay was performed as described40 (link).
Protocol detail
Analysis of α-Dystroglycan Glycosylation
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Anti antibodies
Anti antibody
Antibody
Assay
Enzymes
Glycan
Laminin
Pvdf
Rabbit
Sds page
Sodium acetate
Subunit
Corresponding Organization : University of Iowa
Other organizations : Institute of Biosciences and Bioresources, National Research Council
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Variable analysis
independent variables
- Bgus (0.45 U)
- Xylsa (0.09 U)
- Enriched rabbit α-DG (100 μl of the 150 mM sodium acetate (pH 5.5) solution)
- Protein expression/modification as measured by SDS-PAGE, Western blot with anti-α-DG core antibody (AF6868), anti-α-DG glycan antibodies (IIH6, VIA41), and rabbit polyclonal antibody against the α2 subunit of the DHPR Ca2+ channel
- Laminin binding as measured by laminin overlay assay
- Incubation time (overnight)
- Incubation temperature (65°C)
- Sodium acetate buffer (150 mM, pH 5.5)
- Positive control: Enriched rabbit α-DG (100 μl of the 150 mM sodium acetate (pH 5.5) solution) with no enzymes added
- Negative control: Not explicitly mentioned
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