Lipid binding analysis of 6xHis-tagged B GRAM (Aster-B1–337) was conducted using PIP Strips (Echelon Biosciences), with each spot containing 100 pmol of active lipids. Membranes were blocked with PBS Tween (PBST) solution (supplemented with 3% fatty acid free BSA) for 1 hr at room temperature, and incubated with B GRAM fusion protein in blocking buffer for 1 hr. After three washes, the membranes were blotted with anti-His antibody (Biorad, MCA1396GA). The strip contained 15 different types of lipids. LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; PI, phosphatidylinositol; PI(3)P, phosphatidylinositol 3-phosphate; PI(4)P, phosphatidylinositol 4-phosphate; PI(5)P, phosphatidylinositol 5-phosphate; PE, phosphatidylethanolamine; PC, phosphatidylcholine; S1P, sphingosine-1-phosphate; PI(3,4)P2, phosphatidylinositol 3,4 -phosphate; PI(3,5)P2, phosphatidylinositol 3,5-phosphate; PI(4,5)P2, phosphatidylinositol 4,5-phosphate; PI(3,4,5)P3, phosphatidylinositol 3,4,5-phosphate; PA, phosphatidic acid; PS, phosphatidylserine. Results were confirmed with a FLAG tagged B GRAM Fusion construct.
Mca1396ga
Manufactured by Bio-Rad
The MCA1396GA is a laboratory device manufactured by Bio-Rad. It is designed to perform basic functionality for laboratory applications. Further details on the core function of this product are not available.
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Lab products found in correlation
2 protocols using mca1396ga
1
Lipid Binding Analysis of B GRAM Fusion Protein
2
Western Blot Detection of Trypanosome Proteins
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Western blot analysis was performed as previously described (Salmon et al., 2012a (link)) with modifications for detection of CARP1. Briefly, lysates of 3-5×106 trypanosomes were separated on 10% polyacrylamide gels, transferred to a PVDF membrane via semi-dry blotting and blocked with Kem-En-Tec synthetic blocking buffer for 1 h at room temperature. The blots were incubated with primary antibodies (rabbit anti-CARP1, 1:1000; mouse anti-PFR-A/C [(Kohl et al., 1999 (link)), 1:2000; PFR, paraflagellar rod protein)] overnight at 4°C, followed by secondary antibody (IRDye680LT anti-rabbit and IRDye800CW anti-mouse, LI-COR, both 1:5000) detection for 1.5 h at room temperature. For detection of His-tagged proteins expressed in E. coli, proteins were blotted onto PVDF membranes and blocked with 5% milk for 1 h at room temperature, followed by incubation with mouse anti-His (1:1000, BioRad MCA1396GA) overnight at 4°C and secondary antibody detection with IRDye800CW anti-mouse (1:5000, LI-COR).
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