Spot urine samples were taken weekly (from week 4 to 11) between 2:00 and 3:00 pm and immediately stored at −80°C until assayed. Per week, at least five urine samples were collected from mice of each investigated genotype and sex. Urine creatinine concentrations were determined using an automated analyzer technique (Hitachi, Merck, Darmstadt, Germany). For SDS‐PAGE, urine samples were diluted to a creatinine content of 1.5 mg/dL. Urine proteins were temperature denatured (Thermoblock TB1, Biometra, Germany) and separated using a 12% SDS‐PAGE gel (Protean III, Bio‐Rad, Munich, Germany) together with a broad molecular weight standard (Bio‐Rad) and a mouse albumin standard (Biotrend, Cologne, Germany), as described previously (Herbach et al. 2009 ). Coomassie blue staining of gels was performed according to a standard protocol.
Protean 3
Manufactured by Bio-Rad
Sourced in Germany
The Protean III is a vertical gel electrophoresis system designed for the separation of proteins. It is a reliable and versatile instrument used in various applications within the field of biochemistry and molecular biology.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using protean 3
1
Urine Protein Analysis Protocol
2
PEPC Immunoblotting in Castor Beans
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SDS/PAGE was performed using Bio-Rad Protean III mini-gel (200 V, 50 min) as previously described (Gregory et al., 2009) . Immunoblotting was conducted by electroblotting proteins from gels onto PVDF membranes, and immunoreactive polypeptides were visualized using an alkaline-phosphatase conjugated secondary antibody with chromogenic detection. Immunoblots were probed with anti-(Ricinus communis PEPC) immune serum (anti-PEPC) that had been raised in rabbits against native class-1 PEPC (RcPPC3) fully purified from developing castor beans (Gennidakis et al., 2007) . Anti-(phosphorylation sitespecific)-IgG (anti-pSer11) immunoblots were probed with a polyclonal antibody raised against a synthetic phosphopeptide matching RcPPC3's conserved N-terminal Ser11 phosphorylation domain (L-E-K-L-A-pS-I-D-A-Q-L-R) (Tripodi et al., 2005) . For anti-pSer11 immunoblots the corresponding dephosphopeptide (10 µg/mL) was used to block any nonspecific cross reaction with non-phosphorylated PEPC polypeptides (Tripodi et al., 2005) . All immunoblot results were replicated at least two times, with representative results shown in the various figures.
3
PEPC Immunoblotting in Castor Beans
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SDS/PAGE was performed using Bio-Rad Protean III mini-gel (200 V, 50 min) as previously described (Gregory et al., 2009) . Immunoblotting was conducted by electroblotting proteins from gels onto PVDF membranes, and immunoreactive polypeptides were visualized using an alkaline-phosphatase conjugated secondary antibody with chromogenic detection. Immunoblots were probed with anti-(Ricinus communis PEPC) immune serum (anti-PEPC) that had been raised in rabbits against native class-1 PEPC (RcPPC3) fully purified from developing castor beans (Gennidakis et al., 2007) . Anti-(phosphorylation sitespecific)-IgG (anti-pSer11) immunoblots were probed with a polyclonal antibody raised against a synthetic phosphopeptide matching RcPPC3's conserved N-terminal Ser11 phosphorylation domain (L-E-K-L-A-pS-I-D-A-Q-L-R) (Tripodi et al., 2005) . For anti-pSer11 immunoblots the corresponding dephosphopeptide (10 µg/mL) was used to block any nonspecific cross reaction with non-phosphorylated PEPC polypeptides (Tripodi et al., 2005) . All immunoblot results were replicated at least two times, with representative results shown in the various figures.
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