ABSTRACT
The efficient extraction of proteins of interest from cells and tissues is not always straightforward. Here we demonstrate the differences in extraction of the focal adhesion protein Kindlin-2 from choriocarcinoma cells using NP-40 and RIPA lysis buffer. Furthermore, we demonstrate the use of a more denaturing urea/thiourea lysis buffer for solubilization, by comparing its effectiveness for solubilization of small heat-shock proteins from smooth muscle with the often utilized RIPA lysis buffer. Overall, the results demonstrate the importance of establishing the optimal lysis buffer for specific protein solubilization within the experimental workflow.
Subject(s)
Membrane Proteins/chemistry , Animals , Buffers , Cell Line, Tumor , Electrophoresis, Gel, Two-Dimensional , Electrophoresis, Polyacrylamide Gel , Immunoblotting , Immunoprecipitation , Membrane Proteins/analysis , Membrane Proteins/isolation & purification , Membranes, Artificial , Octoxynol , Polyethylene Glycols/chemistry , SolubilityABSTRACT
The small heat shock protein (HSP) B family of proteins are a group of molecular chaperones that enable tissues to adapt to changes in their local environments during differentiation, stress, or disease conditions. The objective of this research was to characterize the expression of HSPB8 and its cochaperone Bcl2-associated athanogene 3 (BAG3) in nonpregnant (NP) and pregnant rat myometrium during myometrial programming. Rat myometrium was collected from NP and pregnant rats as well as 1 day postpartum (PP) and samples prepared for immunoblot and immunofluorescence analysis. Immunoblot analysis determined that HSPB8 protein expression was significantly elevated at Day (D) 15, D17, and D19 compared to expression at NP and D6, while BAG3 expression was significantly elevated at D15 compared to NP, and D17 compared to NP, D6, D23, and PP time points (P < 0.05). In situ, HSPB8 and BAG3 were predominantly localized to myometrial cells throughout pregnancy, with intense cytoplasmic HSPB8 and BAG3 detection on D15 and D17 in both longitudinal and circular muscle layers. Immunoblot analysis of HSPB8 and BAG3 protein expression in myometrium from unilateral pregnancies also revealed that expression of both proteins was significantly increased at D15 in gravid compared to nongravid horns. Thus, HSPB8 and BAG3 are highly expressed during the synthetic phase of myometrial differentiation marked by initiation of uterine distension and myometrial hypertrophy. HSPB8 and BAG3 could be regulators of the protein quality control required for this process.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Apoptosis Regulatory Proteins/metabolism , Heat-Shock Proteins/metabolism , Myometrium/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Apoptosis Regulatory Proteins/genetics , Female , Gene Expression Regulation/physiology , Heat-Shock Proteins/genetics , Humans , Pregnancy , Rats , Rats, Sprague-DawleyABSTRACT
The efficient extraction of proteins of interest from cells and tissues is not always straightforward. In this process, the use of the optimal lysis buffer for protein solubilization should be considered. Here we demonstrate the use of a urea/thiourea lysis buffer, based on O'Farrell's buffer, and compare its effectiveness for solubilization of proteins from smooth muscle with the often utilized RIPA lysis buffer.
