A simple procedure for the solubilization of NADH-cytochrome b5 reductase

Dey, Arun C.; Rahal, S.; Rimsay, Robert L.; Senciall, Ian R.

doi:10.1016/0003-2697(81)90206-2

Cited by 19 publications

(2 citation statements)

References 12 publications

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“…Sodium phosphate buffer was chosen as extraction buffer because of its ability to extract a wide variety of proteins from tissue in a single extract, which include hydrophilic, hydrophobic, and membrane proteins [21]. Salts [22,23] and glycerol in the extraction buffer were used to increase the solubility of proteins [24], while benzonase was used to reduce the viscosity of the tissue extract by digesting its DNA content [10].…”

Section: Resultsmentioning

confidence: 99%

Analysis of Differentially Expressed Proteins in Colorectal Cancer Using Hydroxyapatite Column and SDS-PAGE

Lim

Gooi²,

Singh³

et al. 2011

Appl Biochem Biotechnol

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Limitation on two dimensional (2D) gel electrophoresis technique causes some proteins to be under presented, especially the extreme acidic, basic, or membrane proteins. To overcome the limitation of 2D electrophoresis, an analysis method was developed for identification of differentially expressed proteins in normal and cancerous colonic tissues using self-pack hydroxyapatite (HA) column. Normal and cancerous colon tissues were homogenized and proteins were extracted using sodium phosphate buffer at pH 6.8. Protein concentration was determined and the proteins were loaded unto the HA column. HA column reduced the complexity of proteins mixture by fractionating the proteins according to their ionic strength. Further protein separation was accomplished by a simple and cost effective sodium dodecyl sulfate-polyacrylamide gel electrophoresis method. The protein bands were subjected to in-gel digestion and protein analysis was performed using electrospray ionization (ESI) ion trap mass spectrometer. There were 17 upregulated proteins and seven downregulated proteins detected with significant differential expression. Some of these proteins were low abundant proteins or proteins with extreme pH that were usually under presented in 2D gel analysis. We have identified brain mitochondrial carrier protein 1, T-cell surface glycoprotein CD1a, SOSS complex subunit B2, and Protein Jade 1 which were previously not detected in 2D gel analysis method.

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Section: Resultsmentioning

confidence: 99%

Analysis of Differentially Expressed Proteins in Colorectal Cancer Using Hydroxyapatite Column and SDS-PAGE

Lim

Gooi²,

Singh³

et al. 2011

Appl Biochem Biotechnol

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“…Placental CRTAase was purified as described earlier [16]. For this purpose, microsomal pellet was solubilized by brief homogenization in 0.1M phosphate buffer, pH 7.4 and 5% sodium cholate using the method of Dey et al with modifications [18]. The clear supernatant obtained after solubilization was dialyzed overnight against 10 mM potassium phosphate buffer (pH 7.4) containing 1 mM EDTA, 1 mM DTT, and 1 mM PMSF.…”

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confidence: 99%

Autoacetylation of Purified Calreticulin Transacetylase Utilizing Acetoxycoumarin as the Acetyl Group Donor

Bansal

Ponnan

Raj

et al. 2008

Appl Biochem Biotechnol

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Our earlier reports documented that calreticulin, a multifunctional Ca2+-binding protein in endoplasmic reticulum lumen, possessed protein acetyltransferase function termed Calreticulin Transacetylase (CRTAase). The autoacetylation of purified human placental CRTAase concomitant with the acetylation of receptor proteins by a model acetoxycoumarin, 7,8-Diacetoxy-4-methylcoumarin, was observed. Here, we have examined the autoacetylation property of CRTAase by immunoblotting and mass spectrometry. Ca2+ was found to inhibit CRTAase activity. The inhibition of both autoacetylation of CRTAase as well as acetylation of the receptor protein was apparent when Ca2+) was included in the reaction mixture as visualized by interaction with anti-acetyl lysine antibody. The acetylation of lysines residues: -48, -62, -64, -153, and -159 in N-domain and -206, -207, -209, and -238 in P-domain of CRTAase were located by high-performance liquid chromatography-electronspray ionization tandem mass spectrometry. Further, computer assisted protein structure modeling studies were undertaken to probe the effect of autoacetylation of CRTAase. Accordingly, the predicted CRTAase 3D model showed that all the loop regions of both N- and P-domain bear the acetylated lysines. Energy minimization of the acetylated residues revealed charge neutralization of lysines due to the N-epsilon-acetylation which may facilitate the interaction of CRTAase with the protein substrate and the subsequent transacetylase action.

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Seminal Findings on a Novel Enzyme: Mechanism of Biochemical Action of 4-Methylcoumarins, Constituents of Medicinal and Edible Plants

et al. 2002

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A simple procedure for the solubilization of NADH-cytochrome b5 reductase

Cited by 19 publications

References 12 publications

Analysis of Differentially Expressed Proteins in Colorectal Cancer Using Hydroxyapatite Column and SDS-PAGE

Analysis of Differentially Expressed Proteins in Colorectal Cancer Using Hydroxyapatite Column and SDS-PAGE

Autoacetylation of Purified Calreticulin Transacetylase Utilizing Acetoxycoumarin as the Acetyl Group Donor

Seminal Findings on a Novel Enzyme: Mechanism of Biochemical Action of 4-Methylcoumarins, Constituents of Medicinal and Edible Plants

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