B cells were isolated from splenocytes from naïve mice as described above, and then the purity was assessed by flow cytometry. Purified B cells (>95%) were lysed with Pierce IP lysis buffer (Thermo Fisher Scientific, 87787). Cell lysates were then quantified and Western blots were performed exactly as previously described (23 (link)). The expression of EWSR1 and β-actin (as a loading control) was detected by using the primary antibodies rabbit anti-EWSR1 at 1:10,000 (Abcam, ab133288) and mouse anti–β-actin at 1:1,000 (Santa Cruz Biotechnology, sc-47778), followed by the secondary antibodies horseradish peroxidase (HRP)–conjugated goat anti-rabbit IgG at 1:5,000 (SouthernBiotech, 4050-05) and goat anti-mouse IgG at 1:5,000 (SouthernBiotech, 1010-05), respectively.
Protocol detail
Quantification of EWSR1 Protein in Purified B Cells
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Pierce IP lysis buffer
ab133288
mouse anti–β-actin
horseradish peroxidase (HRP)–conjugated goat anti-rabbit IgG
goat anti-mouse IgG
Actin
Anti igg
Antibodies
B cells
Buffer
Cell
Ewsr1
Flow cytometry
Goat
Horseradish peroxidase
Mouse
Rabbit
Western blots
Corresponding Organization :
Other organizations : University of Florida, University of Florida Health, Jackson Laboratory
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Variable analysis
independent variables
- Isolation of B cells from splenocytes of naïve mice
- Expression of EWSR1 and β-actin (as a loading control) in purified B cells
- Purity of isolated B cells (>95%)
- Lysis of purified B cells with Pierce IP lysis buffer
- Quantification of cell lysates
- Western blotting performed as previously described (23)
- Positive control: Expression of β-actin as a loading control
- No negative control mentioned
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