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Differential proteome analysis of the preeclamptic placenta using optimized protein extraction.

Centlow M, Hansson SR, Welinder C - J. Biomed. Biotechnol. (2009)

Bottom Line: The removal of glycogen from the samples by centrifugation was crucial for the final proteome maps.Solubilization with urea/CHAPS in combination with dichloromethane/methanol or acidified acetone proved to be the best precipitation procedures.When applied to clinical placenta samples apolipoprotein A1 was found to be accumulated in the preeclamptic placenta, where it may either have a nutritional effect or act as a modifier of signal transduction.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics & Gynecology, Lund University, BMC C14, 22184 Lund, Sweden.

ABSTRACT
The human placenta is a difficult tissue to work with using proteomic technology since it contains large amounts of lipids and glycogen. Both lipids and glycogen are known to interfere with the first step in the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), the isoelectric focusing. In order to gain the best possible protein separation on 2D-PAGE, an optimized sample preparation protocol for placental proteins was developed. Two different buffers, urea/CHAPS and Hepes, were used for solubilization in combination with six different precipitation methods. The removal of glycogen from the samples by centrifugation was crucial for the final proteome maps. Solubilization with urea/CHAPS in combination with dichloromethane/methanol or acidified acetone proved to be the best precipitation procedures. When applied to clinical placenta samples apolipoprotein A1 was found to be accumulated in the preeclamptic placenta, where it may either have a nutritional effect or act as a modifier of signal transduction.

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Two-dimensional separation of precipitated proteins (100 μg) in PE placenta solubilized in (a) urea/CHAPS and (b) Hepes buffer. Precipitation procedures (1) dichloromethane/methanol, (2) TCA, (3) TCA followed by ethanol wash, (4) acetone, (5) acidified acetone, and (6) ethanol. The gels were stained with silver. Arrows on lower right image represent directions for increasing Mw and pI. Directions are the same for all gels.
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fig2: Two-dimensional separation of precipitated proteins (100 μg) in PE placenta solubilized in (a) urea/CHAPS and (b) Hepes buffer. Precipitation procedures (1) dichloromethane/methanol, (2) TCA, (3) TCA followed by ethanol wash, (4) acetone, (5) acidified acetone, and (6) ethanol. The gels were stained with silver. Arrows on lower right image represent directions for increasing Mw and pI. Directions are the same for all gels.

Mentions: Samples solubilized with urea/CHAPS generally resulted in more protein spots and less horizontal streaking, regardless of precipitation procedure (Figure 2). The investigation of whether freezing of solubilized and centrifuged samples influenced the results, both fresh and frozen samples, was analyzed using 2D-PAGE. No major differences could be detected in the protein expression profiles for any of the different solubilization or precipitation methods (data not shown). An increased number of low molecular weight proteins were obtained with samples solubilized in urea/CHAPS precipitated with acidified acetone (Figure 2, A5).


Differential proteome analysis of the preeclamptic placenta using optimized protein extraction.

Centlow M, Hansson SR, Welinder C - J. Biomed. Biotechnol. (2009)

Two-dimensional separation of precipitated proteins (100 μg) in PE placenta solubilized in (a) urea/CHAPS and (b) Hepes buffer. Precipitation procedures (1) dichloromethane/methanol, (2) TCA, (3) TCA followed by ethanol wash, (4) acetone, (5) acidified acetone, and (6) ethanol. The gels were stained with silver. Arrows on lower right image represent directions for increasing Mw and pI. Directions are the same for all gels.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2742651&req=5

fig2: Two-dimensional separation of precipitated proteins (100 μg) in PE placenta solubilized in (a) urea/CHAPS and (b) Hepes buffer. Precipitation procedures (1) dichloromethane/methanol, (2) TCA, (3) TCA followed by ethanol wash, (4) acetone, (5) acidified acetone, and (6) ethanol. The gels were stained with silver. Arrows on lower right image represent directions for increasing Mw and pI. Directions are the same for all gels.
Mentions: Samples solubilized with urea/CHAPS generally resulted in more protein spots and less horizontal streaking, regardless of precipitation procedure (Figure 2). The investigation of whether freezing of solubilized and centrifuged samples influenced the results, both fresh and frozen samples, was analyzed using 2D-PAGE. No major differences could be detected in the protein expression profiles for any of the different solubilization or precipitation methods (data not shown). An increased number of low molecular weight proteins were obtained with samples solubilized in urea/CHAPS precipitated with acidified acetone (Figure 2, A5).

Bottom Line: The removal of glycogen from the samples by centrifugation was crucial for the final proteome maps.Solubilization with urea/CHAPS in combination with dichloromethane/methanol or acidified acetone proved to be the best precipitation procedures.When applied to clinical placenta samples apolipoprotein A1 was found to be accumulated in the preeclamptic placenta, where it may either have a nutritional effect or act as a modifier of signal transduction.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics & Gynecology, Lund University, BMC C14, 22184 Lund, Sweden.

ABSTRACT
The human placenta is a difficult tissue to work with using proteomic technology since it contains large amounts of lipids and glycogen. Both lipids and glycogen are known to interfere with the first step in the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), the isoelectric focusing. In order to gain the best possible protein separation on 2D-PAGE, an optimized sample preparation protocol for placental proteins was developed. Two different buffers, urea/CHAPS and Hepes, were used for solubilization in combination with six different precipitation methods. The removal of glycogen from the samples by centrifugation was crucial for the final proteome maps. Solubilization with urea/CHAPS in combination with dichloromethane/methanol or acidified acetone proved to be the best precipitation procedures. When applied to clinical placenta samples apolipoprotein A1 was found to be accumulated in the preeclamptic placenta, where it may either have a nutritional effect or act as a modifier of signal transduction.

Show MeSH
Related in: MedlinePlus