Lectins for Western Blot Detection of Glycoproteins

Six peroxidase-labeled lectins (HRP-lectins) were optimized for the detection of twelve different glycoproteins utilizing chemiluminescent peroxidase detection.

Protocol
  • Glycoprotein sample size: 500ng
  • Lectin Concentration: 0.1ug/ml

1. Load at 500ng of protein per lane
2. Run 4-20% gradient Bis-Tris SDS page gel
3. Transfer gel to a PVDF membrane
4. Block membrane for 1 hour at room temperature with RIPA buffer (Sigma Prod. No. R0278 Sigma)
5. Incubate HRP-lectin at 0.1ug/ml with RIPA buffer for 2 hours at room temperature
6. Wash membrane 5 x 5 minutes with 25ml RIPA buffer
9. Detect using chemiluminescent substrate (Sigma Prod. No. CPS1-120)

Lane assignments for glycoprotein samples analyzed
1. Urokinase from human urine (Sigma U0633)
2. Factor IX Human (Sigma F0806)
3. Complement component C1q from human serum (Sigma C1740)
4. α1-Antitrypsin from human plasma (Sigma A3106)
5. Fetuin (Sigma F2379)
6. Invertase glycoprotein Standard (Sigma I0408)
7. IgG1 Monoclonal Anti-Flag M2 Ab produced in mouse (Sigma F3165)
8. IgG2a Monoclonal Anti-AMY1A Ab produced in mouse (Sigma WH0000276M4)
9. Recombinant Human t-Plasminogen Activator/tPA
10. Epidermal growth factor receptor human (Sigma E3641)
11. Apolipoprotein C-III from human plasma (Sigma A3106)
12. Thrombospondin from human platelets
Western Blot of twelve glycoproteins using HRP conjugated Arachis hypogea lectin specific f
Western Blot of twelve glycoproteins using HRP conjugated Bandeiraea simplicifolia
Western Blot of twelve glycoproteins using HRP conjugated Canavalia ensiformis lectin speci
Western Blot of twelve glycoproteins using HRP conjugated Glycine max lectin specific for g
Western Blot of twelve glycoproteins using HRP conjugated Triticum vulgaris lectin specific
Western Blot of twelve glycoproteins using HRP conjugated Ulex europaeus lectin specific fo

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L6397 Concanavalin A from Canavalia ensiformis (Jack bean) peroxidase conjugate, lyophilized powder
L7759 Lectin from Arachis hypogaea (peanut) peroxidase conjugate, lyophilized powder
L5391 Lectin from Bandeiraea simplicifolia (Griffonia simplicifolia) Isolectin B4 (BSI-B4), peroxidase conjugate, lyophilized powder
L2650 Lectin from Glycine max (soybean) peroxidase conjugate, lyophilized powder
L3892 Lectin from Triticum vulgaris (wheat) peroxidase conjugate, lyophilized powder
L8146 Lectin from Ulex europaeus (gorse, furze) peroxidase conjugate, lyophilized powder