The antibodies used are listed in Supplemental Table S2. For generation of an antibody to the IFT46 C-terminus, PCR primer pair IFT46-29 and IFT46-14 was used to amplify a 0.7-kb sequence encoding IFT46 aa 105–344 from a cloned IFT46 cDNA (Hou et al., 2007 (link)). The PCR product was digested with BamHI and inserted into the BamHI site of pMAL-cR1 (New England Biolabs, Beverly, MA). Expression of this construct in Escherichia coli produced a protein in which IFT46 aa 105–344 were fused to maltose-binding protein. The fusion protein was purified by amylase affinity chromatography, and antibodies were raised against the fusion protein in guinea pigs (Covance Research Products, Denver, PA).
Protocol detail
Generation of IFT46 C-terminus Antibody
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A protein
Aa 344
Affinity chromatography
Amylase
Antibodies
Antibody
Cdna
Escherichia coli
Guinea pigs
Maltose binding protein
Primer
Protein
Corresponding Organization : University of Massachusetts Chan Medical School
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- PCR primer pair IFT46-29 and IFT46-14 used to amplify a 0.7-kb sequence encoding IFT46 aa 105–344 from a cloned IFT46 cDNA
- Antibodies raised against the fusion protein in guinea pigs
- Expression of the construct in Escherichia coli to produce a protein in which IFT46 aa 105–344 were fused to maltose-binding protein
- Purification of the fusion protein by amylase affinity chromatography
- No positive or negative controls were explicitly mentioned.
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