Journal: BioTechniques
Article Title: Quantitative recovery of immunoreactive proteins from clinical samples following RNA and DNA isolation.
doi: 10.2144/03353bm02
Figure Lengend Snippet: Figure 1. Comparison of yield and immunoreactivity of proteins from chorionic villous samples extracted and solubilized with different reagents. (A) Glycogen, at a high pH, and sarkosyl (SK) increased the solubility of proteins in TRIZOL protein pellets from villous tissue (39-week pregnancy). Protein pellets, precipitated from TRIZOL supernatants in the absence of glycogen, were extracted with 50 mM Tris-Cl buffer at the pH indicated from the equivalent of 2.5 mg tis- sue (lanes 2–9), from 1–10 mg tissue precipitated in the presence of glycogen (lanes 11–14; in lane 11, 2.5 mg tissue equivalent were loaded and, in lane 12, 2 mg tissue equivalent were loaded), and from 2 mg tissue with or without sarkosyl at pH 6.8 (lanes 16 and 17) and at pH 8.8 (lanes 18 and 19). Protein concentrations from six different experiments were measured, and the mean (x– ± SEM) relative increase in recovery at pH 7.5, 8.0, and 8.8, compared to that at pH 6.8, are shown (lanes 2–9). Patient samples were villous tissues from two 39-week (39- and 38.4-week, caesarean delivery), two 12-week (12- and 12.1-week, voluntary termi- nation of pregnancy) pregnancies, and fetal brain (13.1-week with Down syndrome). (B) Lanes 2–5, proteins were directly extracted from 50 mg villous tissue (39-week pregnancy) at pH 8.0, tissue protein extraction reagent (T-PER), and from TRIZOL pellets (lanes 6 and 7). (C) Villous proteins (38.4-week pregnancy) extracted directly (lanes 1–4) at conditions indicated were resolved in 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels, electrotransferred to Immobilon polyvinylidene difluoride (PVDF) membrane, immunostained [upper panel; antibody (Ab)] with affinity-purified polyclonal goat anti-human interferon γ recep- tor (IFNGR) 2, and membranes were then stained with 0.25% Coomassie blue (lower panel; stain). (D) Similar experiment to panel A (lanes 2–9), except that vil- lous tissue was from a 12-week pregnancy and the proteins transferred to a PVDF membrane were first immunoreacted with anti-human IFNGR 2 antibody, and, subsequently, the membrane was stripped and reprobed with β-actin antibody. Fold increase in staining at different pHs compared to that at pH 6.8 is shown below the bands; lanes 9 and 10, proteins were extracted at pH 8.8, and fold-increase by glycogen is shown for IFNGR 2 and β-actin. (E) Similar experiment (villous tissue from a 12.1-week pregnancy) to panel D, except it was first immunostained with anti-human leukemia inhibitory factor-soluble receptor α (LIF-sRα) and subsequently with β-actin. (F) Villous proteins (38.4-week pregnancy) were extracted at pH 6.8 (lanes 1 and 2), pH 8.0 (lanes 3 and 4), and pH 8.8 (lanes 5 and 6) in the presence and absence of sarkosyl. (G) RNA analysis from samples used for protein extraction; lanes 1 and 2, 5 µg total RNA from experiments shown in panels A–C and F (two 39-week villous tissue) were resolved on agarose gel, which demonstrated the quality of 28S and 18S ribosomal RNAs; the cDNA pre- pared from 12-week (Figure 1D) and 39-week pregnancies (A) were used to amplify IFNGR1 (forward, 5′-GAATGAACGGAAGTGAGATCCA-3′ and reverse, 5′-CCTGGAACTGTTGCTGGAGA-3′), 8.0 (forward, 5′-TCAGGGATAGCCCCCATCTAT-3′ and reverse, 5′-AACCCTTTGCCACTACATCAATTT-3′), and 3.1 kb (forward, 5′-CCGCTGAAAGAGGGGGAA-3′ and reverse, 5′-TAGAGGTTGTGCAGCTGAGA-3′) Syncytin, and β-actin (forward, 5′-CTTCTACAAT- GAGCTGGGTG-3′ and reverse, 5′-TCATGAGGTAGTCAGTCAGG-3′) mRNAs alone or together (multiplex), as previously described (10).
Article Snippet: The effects of pH, glycogen, and sarkosyl on the retrieval of proteins from TRIZOL pellets were verified (Figure 1, C–F) by the Western transfer of proteins to ImmobilonTM polyvinylidene difluoride (PVDF) membranes (Millipore, Watford, UK), followed by detection using antibodies raised against various epitopes [human interferon γ receptors (IFNGR) 1 and 2; human leukemia inhibitory factor-soluble receptor α (LIF sRα) (R&D Systems, Oxford, UK); β-actin, clone AC-15 (Sigma, Poole, Dorset, UK)].
Techniques: Comparison, Solubility, Protein Extraction, Membrane, Affinity Purification, Staining, Agarose Gel Electrophoresis, Multiplex Assay
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![Figure 1. Comparison of yield and immunoreactivity of proteins from chorionic villous samples extracted and solubilized with different reagents. (A) Glycogen, at a high pH, and sarkosyl (SK) increased the solubility of proteins in TRIZOL protein pellets from villous tissue (39-week pregnancy). Protein pellets, precipitated from TRIZOL supernatants in the absence of glycogen, were extracted with 50 mM Tris-Cl buffer at the pH indicated from the equivalent of 2.5 mg tis- sue (lanes 2–9), from 1–10 mg tissue precipitated in the presence of glycogen (lanes 11–14; in lane 11, 2.5 mg tissue equivalent were loaded and, in lane 12, 2 mg tissue equivalent were loaded), and from 2 mg tissue with or without sarkosyl at pH 6.8 (lanes 16 and 17) and at pH 8.8 (lanes 18 and 19). Protein concentrations from six different experiments were measured, and the mean (x– ± SEM) relative increase in recovery at pH 7.5, 8.0, and 8.8, compared to that at pH 6.8, are shown (lanes 2–9). Patient samples were villous tissues from two 39-week (39- and 38.4-week, caesarean delivery), two 12-week (12- and 12.1-week, voluntary termi- nation of pregnancy) pregnancies, and fetal brain (13.1-week with Down syndrome). (B) Lanes 2–5, proteins were directly extracted from 50 mg villous tissue (39-week pregnancy) at pH 8.0, tissue protein extraction reagent (T-PER), and from TRIZOL pellets (lanes 6 and 7). (C) Villous proteins (38.4-week pregnancy) extracted directly (lanes 1–4) at conditions indicated were resolved in 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels, electrotransferred to Immobilon polyvinylidene difluoride (PVDF) membrane, immunostained [upper panel; antibody (Ab)] with affinity-purified polyclonal goat anti-human interferon γ recep- tor (IFNGR) 2, and membranes were then stained with 0.25% Coomassie blue (lower panel; stain). (D) Similar experiment to panel A (lanes 2–9), except that vil- lous tissue was from a 12-week pregnancy and the proteins transferred to a PVDF membrane were first immunoreacted with anti-human IFNGR 2 antibody, and, subsequently, the membrane was stripped and reprobed with β-actin antibody. Fold increase in staining at different pHs compared to that at pH 6.8 is shown below the bands; lanes 9 and 10, proteins were extracted at pH 8.8, and fold-increase by glycogen is shown for IFNGR 2 and β-actin. (E) Similar experiment (villous tissue from a 12.1-week pregnancy) to panel D, except it was first immunostained with anti-human leukemia <t>inhibitory</t> factor-soluble <t>receptor</t> <t>α</t> <t>(LIF-sRα)</t> and subsequently with β-actin. (F) Villous proteins (38.4-week pregnancy) were extracted at pH 6.8 (lanes 1 and 2), pH 8.0 (lanes 3 and 4), and pH 8.8 (lanes 5 and 6) in the presence and absence of sarkosyl. (G) RNA analysis from samples used for protein extraction; lanes 1 and 2, 5 µg total RNA from experiments shown in panels A–C and F (two 39-week villous tissue) were resolved on agarose gel, which demonstrated the quality of 28S and 18S ribosomal RNAs; the cDNA pre- pared from 12-week (Figure 1D) and 39-week pregnancies (A) were used to amplify IFNGR1 (forward, 5′-GAATGAACGGAAGTGAGATCCA-3′ and reverse, 5′-CCTGGAACTGTTGCTGGAGA-3′), 8.0 (forward, 5′-TCAGGGATAGCCCCCATCTAT-3′ and reverse, 5′-AACCCTTTGCCACTACATCAATTT-3′), and 3.1 kb (forward, 5′-CCGCTGAAAGAGGGGGAA-3′ and reverse, 5′-TAGAGGTTGTGCAGCTGAGA-3′) Syncytin, and β-actin (forward, 5′-CTTCTACAAT- GAGCTGGGTG-3′ and reverse, 5′-TCATGAGGTAGTCAGTCAGG-3′) mRNAs alone or together (multiplex), as previously described (10).](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_3546/pm14513546/pm14513546__page2_image1.jpg)