S100B, Synaptic Systems, 287 003
What do reviewers generally think about 287 003 from Synaptic Systems?
Researchers using S100B, Synaptic Systems, 287 003 on rat astrocytes report poor performance. One reviewer points out that Western blotting of primary rat astrocyte lysates gave no signal, despite standard transfer, blocking, 1:1000 primary overnight, and HRP secondary detection. Another reviewer found strong background in immunofluorescence of fixed, differentiated rat astrocytes, using 0.002 mg/mL primary antibody and Alexa Fluor 488 secondary. The consensus is that the antibody failed to provide useful specific signal in these applications.
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Review of: S100B, Synaptic Systems, 287 003 by Andrea E Toth
No signal in WB.
Sample: primary rat astrocytes
Cells were lysed in ExB lysis buffer (150 mM NaCl, 20 mM MgCl2, 20 mM CaCl2, 100 mM HEPES, 1% TritonX-100, complete protease inhibitor). 2.7 μg of each protein sample was loaded on a 4–12% polyacrylamide gel (Genescript) and subsequently transferred to nitrocellulose membrane. Membranes were blocked in 5% skimmed milk, 0.01 M Tris–HCl, 0.15 M NaCl, and 0.1% Tween 20, pH 7.6 in buffer solution at room temperature (RT). Primary antibodies (1:1000) were applied overnight at 4 °C. On the following day, membranes were incubated with HRP-conjugated secondary antibodies (1:2000) for 1 h at RT. Specific band size was detected with ECL (GE Healthcare, Brøndby, Denmark) according to the manufacturer’s recommendations and visualized using LAS 4000 (Fujifilm).
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0 not usefulReview of: S100B, Synaptic Systems, 287 003 by Andrea E Toth
Cells: rat astrocytes
cultured for 4 week after isolation
and differentiated with cAMP and hydrocortisone treatment for 1 day
Fixation: 4% paraformaldehyde
Permeabilization: 0.2% triton X-100 in PBS for 10 min
Blocking: 2% bovine serum albumin (BSA) in PBS for 20 min
Primary antibody: 0.002 mg/mL in 2% BSA, 0.05% triton X-100 in PBS 1h RT
Secondary antibody: 1:1000 donkey anti-rabbit IgG Alexa Fluor 488 (Invitrogen; A21206) in 2% BSA, 0.05% triton X-100 in PBS 1h RT
Evaluation: strong back grand
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