I am experiencing an issue where my target protein is not detected in the extract from SH-SY5Y cells. I am wondering if this could be related to my A/G magnetic beads or another aspect of my protocol. The results are shown in the attached image.
Here’s a brief overview of my procedure:
Given these steps, could anyone provide insights into why my target protein might not be present in the extract? Are there specific factors in my protocol that I should consider adjusting, or could it indeed be an issue with the magnetic beads themselves? Any guidance or suggestions would be greatly appreciated!
Marine Ma , the possibility of a magnetic bead problem is low, and SH-SY5Y doesn't seem to express your target protein.
Your input sample (not IP) doesn't show the 35kd band when run directly on Western blotting.
Screening magnetic bead problems is as follows:
A/G magnetic beads are compatible with the antibody type: Protein A and Protein G have different affinities for IgG of different species and subtypes. If the antibody is mouse IgG1, which has poor affinity for Protein A, it is recommended to use Protein G or mixed A/G magnetic beads.
Are the magnetic beads thoroughly washed? Residual sodium azide (NaN₃) or storage buffer may affect binding; ensure multiple PBS washes.
Bead freshness and activity: Long-term storage or repeated freeze-thaw cycles can reduce binding capacity; use fresh beads.
Next, there's the issue of the antibody.
You used 6.25 μmol/ml, but the normal antibody concentration is typically 1–10 μg/ml (note, μg/ml).
The answer to this question comes from MedChemExpress (MCE) Technical Support.
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