Comparative analysis of insect cell-expressed rAamIGFBP-rP1 and human recombinant IGFBP-rP1 (Fitzgerald, Acton, MA, USA) trypsin digestion was performed. Around 0.5 µg of protein in 20 µL of PBS was subjected to digestion with bovine trypsin in the final concentrations of 5–100 µg/mL for 15 min at 37°C. Products of digestion were separated on 12.5% SDS-PAGE and visualized with standard Coomassie brilliant blue staining. In addition, western blot analysis was performed with digested samples using HRP-labeled anti C-terminal His-tag antibody (Life Technologies), as well as previously generated antibodies to A. americanum tick saliva proteins (Mulenga et al., 2013b (link)), to detect separated fragments. Positive signal was detected by using BioFX chemiluminescent reagent (SurModics).
Protocol detail
Comparative Trypsin Digestion of rAamIGFBP-rP1
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Antibodies
Antibody
Bovine
Cell
Coomassie brilliant blue
Digestion
Human
Igfbp rp1
Insect
Protein
Saliva
Sds page
Tick proteins
Trypsin
Western blot analysis
Corresponding Organization :
Other organizations : Texas A&M University
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Variable analysis
independent variables
- Trypsin concentration (5-100 µg/mL)
- Digestion products of rAamIGFBP-rP1 and human recombinant IGFBP-rP1 separated on 12.5% SDS-PAGE
- Detection of separated fragments using Western blot analysis with HRP-labeled anti C-terminal His-tag antibody and previously generated antibodies to A. americanum tick saliva proteins
- Protein amount (0.5 µg)
- Buffer (PBS)
- Digestion time (15 min)
- Digestion temperature (37°C)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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