Organoids or crypt-enriched fractions from mouse ileum were concentrated
by centrifugation, resuspended in 30% acetic acid, and sonicated. After
incubation overnight at 4 °C with agitation, samples were diluted 3-fold
with water. Insoluble material was removed by centrifugation at 100,000
× g for 2 h at 4 °C, protein concentrations of the supernatants
were determined by Bio-Rad Protein Assay (Bio-Rad), and equivalent amounts of
each sample were lyophilized. Lyophilized samples were dissolved in 5%
acetic acid and separated by 17% AU-PAGE14 (link). Folded Crp23 was created from a
synthesized 80% pure linear peptide (CPC Scientific) by the same
procedure as previously reported for the α-defensin HD540 (link). Proteins were visualized with
SYPRO Ruby (Life Technologies). Gels were imaged using a Typhoon 9400 variable
mode imager (GE Healthcare). For western blot, samples were separated by
12.5% AU-PAGE and semi-dry transferred to nitrocellulose membranes.
Membranes were immediately fixed in glutaraldahyde and blocked in 5%
milk, before overnight RT incubation in rabbit anti-HD5 antibody (kind gift from
Edith Porter12 (link)) at a 1:1000
dilution. Membranes were incubated in goat-anti-rabbit Alexa Fluor 488
(Invitrogen) and imaged using a Typhoon 9400 variable mode imager (GE
Healthcare).
by centrifugation, resuspended in 30% acetic acid, and sonicated. After
incubation overnight at 4 °C with agitation, samples were diluted 3-fold
with water. Insoluble material was removed by centrifugation at 100,000
× g for 2 h at 4 °C, protein concentrations of the supernatants
were determined by Bio-Rad Protein Assay (Bio-Rad), and equivalent amounts of
each sample were lyophilized. Lyophilized samples were dissolved in 5%
acetic acid and separated by 17% AU-PAGE14 (link). Folded Crp23 was created from a
synthesized 80% pure linear peptide (CPC Scientific) by the same
procedure as previously reported for the α-defensin HD540 (link). Proteins were visualized with
SYPRO Ruby (Life Technologies). Gels were imaged using a Typhoon 9400 variable
mode imager (GE Healthcare). For western blot, samples were separated by
12.5% AU-PAGE and semi-dry transferred to nitrocellulose membranes.
Membranes were immediately fixed in glutaraldahyde and blocked in 5%
milk, before overnight RT incubation in rabbit anti-HD5 antibody (kind gift from
Edith Porter12 (link)) at a 1:1000
dilution. Membranes were incubated in goat-anti-rabbit Alexa Fluor 488
(Invitrogen) and imaged using a Typhoon 9400 variable mode imager (GE
Healthcare).