PDE3A and PKG-Iα constructs were transiently transfected in HEK293 cells for 48 h. PDE3A was immunoprecipitated from the cell lysates as described previously. The protein was resolved using 4 to 20% Tris–glycine SDS-PAGE and visualized by colloidal Coomassie stain (Bio-Rad Laboratories). The band corresponding to PDE3A (approximately 130 kDa) was excised and destained with 100 mM ammonium bicarbonate/50% (v/v) acetonitrile. Protein in gel was subjected to reduction and alkylation of cysteine residues before overnight in-gel digestion with chymotrypsin in 100 mM Tris–HCl containing 10 mM CaCl2. The peptides were extracted with 0.1% TFA, 60% acetonitrile, and evaporated to near dryness. MS analysis of PDE3A was then performed as described (59 (link)). All spectra were taken on an ABSciex 5800 MALDI-TOF mass spectrometer in positive reflector mode (10 kV) with a matrix of cyano-4-hydroxycinnamic acid. At least 1000 laser shots were averaged to get each spectrum. The masses were calibrated to known peptide standards. The MS spectra were analyzed in the ProteinPilot software package (AB Sciex).
Colloidal coomassie stain
Manufactured by Bio-Rad
Colloidal Coomassie stain is a protein detection solution used in gel electrophoresis. It is designed to stain proteins in polyacrylamide gels.
Automatically generated - may contain errors
3 protocols using colloidal coomassie stain
1
PDE3A Immunoprecipitation and Proteomic Analysis
2
Proteomic Analysis of Cell Fractions
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Protein concentration of fractions was determined by Bradford assay (Bio-Rad #5000006). 10 µg of the total cell homogenates and SL fraction F2 (each corresponding to a biological triplicate) were used for proteomic analysis. Briefly, samples were mixed with SDS–PAGE sample buffer and denatured before 1-dimensional electrophoresis (1-DE) through an Invitrogen precast 10% NuPAGE Bis-Tris gel with corresponding NuPAGE running buffer at 80 V for 1 h. The gel was washed with Milli-Q H2O and fixed with 40% ethanol, and 10% acetic acid in Milli-Q H2O for 15 min with gentle rocking. After a series of Milli-Q H2O washes, the gel was stained using a pre-prepared Bio-Rad Colloidal Coomassie stain (#1610803) and then de-stained with Milli-Q H2O. The gel lanes were then divided into 10 fractions. The 10 gel fractions were further divided into 1 mm square cubes, further de-stained, and then digested in-gel with trypsin to extract peptides according to the methods of Shevchenko et al.65 (link). Peptides were dried using a Speed-Vac and analyzed by mass spectrometry.
3
Protein Profiling of Cell Fractions
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Protein concentration of fractions was determined by Bradford assay (Bio-Rad #5000006). 10 µg of the total cell homogenates and SL fraction F2 (each corresponding to a biological triplicate) were used for proteomic analysis. Briefly, samples were mixed with SDS-PAGE sample buffer and denatured before 1-dimensional electrophoresis (1-DE) through an
Invitrogen precast 10% NuPAGE Bis-Tris gel with corresponding NuPAGE running buffer at 80V for 1h. The gel was washed with Milli-Q H2O and fixed with 40% ethanol, 10% acetic acid in Milli-Q H2O for 15min with gentle rocking. After a series of Milli-Q H2O washes, the gel was stained using a pre-prepared Bio-Rad Colloidal Coomassie stain (#1610803) and then destained with Milli-Q H2O. The gel lanes were then divided into ten fractions. The ten gel fractions were further divided into 1mm square cubes, further de-stained and then digested in-gel with trypsin to extract peptides according to the methods of (Shevchenko et al., 2006) .
Peptides were dried using a Speed-Vac and analyzed by mass spectrometry.
Invitrogen precast 10% NuPAGE Bis-Tris gel with corresponding NuPAGE running buffer at 80V for 1h. The gel was washed with Milli-Q H2O and fixed with 40% ethanol, 10% acetic acid in Milli-Q H2O for 15min with gentle rocking. After a series of Milli-Q H2O washes, the gel was stained using a pre-prepared Bio-Rad Colloidal Coomassie stain (#1610803) and then destained with Milli-Q H2O. The gel lanes were then divided into ten fractions. The ten gel fractions were further divided into 1mm square cubes, further de-stained and then digested in-gel with trypsin to extract peptides according to the methods of (Shevchenko et al., 2006) .
Peptides were dried using a Speed-Vac and analyzed by mass spectrometry.
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