**2.5. Protein quantification**

Healthcare, USA, 17-1314-01), Tris (Bio-Rad, USA, 1610716), biolyte 3/10 ampholyte (Bio-Rad, USA, 1632094), dithiothreitol (DTT, GE Healthcare, USA, 17-1318-02), hydrochloric acid solution (HCl, Fisher Chemical, UK, SA48-500), ethylenediaminetetraacetic acid (EDTA, Sigma, USA, E9884), potassium chloride (KCl, Sigma-Aldrich, USA, P9541), sucrose (Fisher Reagent, USA, S25590), methanol (Fisher Reagent, USA, A4521), dichloromethane (DCM, Fisher Reagent, USA, AC326600025), lithium chloride (LiCl, Sigma-Aldrich, USA, 13013), 2D quant kit (GE Healthcare, USA, 80-6483-56), glycerol (Sigma-Aldrich, USA, G5516), sodium dodecyl sulfate (SDS, Bio-Rad, USA, 1610302), bromophenol blue (Bio-Rad, USA, 1610404), pre-stained BLUeye (Gene Direx, USA, PM007-0500), acrylamide/bis mixed solution (37.5:1) (Nacalai tecque, Japan, 06121), wide-range gel preparation buffer (Nacalai tecque, Japan, 07831-94), N,N,N′,N′-tetramethylethylenediamine (TEMED, GE Healthcare, USA, 17-1312-01), ammonium persulfate (APS, Sigma-Aldrich, USA, 09913), agarose NA (GE Healthcare, USA, 17-0554- 01), glycine (Bio-rad, USA, 1610718), DNase I (New England Biolabs, UK, M0303 L), immobiline dry strip pH 3-10 non-linear, 13 cm (GE Healthcare, USA, 17-6001-15), dry strip cover fluid (GE Healthcare, USA, 17-1335-01), iodoacetamide (IAA, Bio-Rad, USA, 1632109), formaldehyde solution (Sigma-Aldrich, USA, F8775), silver nitrate (Sigma-Aldrich, USA, 209139), sodium carbonate (Sigma-Aldrich, USA, S7795), sodium thiosulfate pentahydrate (Sigma-Aldrich, USA, 217247), glacial acetic acid (Sigma-Aldrich, USA, 320099), ethanol (EtOH, Fisher Chemical, USA, A955-4), phosphoric acid (Fisher Chemical, USA, A260-500), ammonium sulfate (Fisher Chemical, USA, A702-500), Coomassie brilliant blue G-250 (Sigma, UK, 27815), acetonitrile (Fisher Chemical, UK, A998-212), acetic acid (Fisher Chemical, USA, A35-500), ammonium hydrogen citrate (Merck, UK, 101154), trifluoroacetic acid (TFA, Sigma-Aldrich, UK, 302031),

*P* < 0.05

**Characteristics Case (***n* **= 5) Control (***n* **= 10) P value** Estimated gestational age (weeks) 35.4 ± 0.89 38.5 ± 0.53 – Maternal age (years) 33.2 ± 5.89 32.7 ± 5.93 NS Parity 3 (1–6) 4 (1–6) NS Fetus weight (kg) 2.32 ± 0.40 3.22 ± 0.25 0.02\* Placenta weight (g) 384 ± 20.74 488.5 ± 11.56 <0.002\* NS, not significant; data are presented as means ± SEM, except parity data are presented as median (range).\*

Milli-q water (Milli-Q Integral 3 water purification system, Merck Millipore, USA) was used

To improve the homogeneity and comparability, placentas selected in this study were collected from pregnant women who were experiencing sPTL-IM or sTL-IM with prior history

and α-cyano-4-hydroxycinnamic acid (Sigma-Aldrich, UK, C8982).

**Table 1.** Demographic and clinical details of women participated in this study.

in preparing all the chemical solutions required.

when compared to the other group.

4 Electrophoresis - Life Sciences Practical Applications

**2.3. Tissue collection, processing, and storage**

of spontaneous PTL-IM among Malay ethnicity only.

The total placentas protein concentration of the purified placentas tissue lysates from both case and control (maternal and fetal sides) were quantified by 2D Quant kit (GE Healthcare) according to the manufacturer's instruction. Bovine serum albumin was used as a standard throughout all the independent experiments.
