Purification:
Article Title: Interaction of serotonin transporter and TGFB1 facilitates histone serotonylation-mediated synaptic plasticity following environmental enrichment in stressed mouse Mus booduga
Article Snippet: The membrane was then documented and the intensity of specific bands was measured using ChemiDoc XRS Image Lab-2 (Bio-Rad Laboratories, Inc., USA). .. The purified protein sample (200 μg; Cat # 1632130, 2-D cleanup kit- Bio-Rad Laboratories, Inc., USA) was passively rehydrated on IPG Strips (pH: 3–10, 7 cm, ReadyStripTM IPG strip, Bio-Rad Laboratories, Inc.) for 16 h. Then, IPG strips were subjected to isoelectric focusing (IEF; Bio-Rad PROTEAN ® IEF cell) in four steps: (i) a rapid phase – 500 V (15 min), (ii) gradual phase – 4000 V (1 h), (iii) rapid phase – 4000 V (15000 V/h), (iv) a hold step at 500 V. After completion of IEF, the strips were reduced and alkylated using equilibration buffers I and II (10 min/each) (ReadyPrepTM 2-D starter kit). .. Proteins from the IPG strip were separated in the second dimension (10% TGX stain free acrylamide kit, Cat# 1610183, Bio-Rad Laboratories, Inc.) and subjected to western blot analysis using anti-SERT rabbit polyclonal antibody (Cat # AB9726, 1:2000; Merck Millipore), anti- TGFB1rabbit polyclonal antibody (Cat # 3711, 1:1000; Cell Signaling Technology), and secondary antibody goat anti-rabbit IgG ALP (Cat# 621100180011730; 1:5000; GeNeiTM), as mentioned earlier.
Article Title: P1PK blood group antigens in birds: implications for veterinary blood transfusion, human and pet health
Article Snippet: Lyophilized pigeons RBC membranes (60 mg) were suspended in 500 μl lysis buffer (250 mM Tris–HCl pH 8.0, 50 mM NaCl, and 1X proteinases inhibitor cocktail) (Thermo Fisher Scientific, USA), passed through a 22G × 1 1⁄2 needle 20 times, and centrifuged for 15 min with a maximum speed (15,000 × g) of 4 0 C. The supernatant (protein lysate) was collected for additional purification before isoelectric focusing. (IEF). .. The protein lysate (100 μl) was purified before the first dimension run of 2D electrophoresis using ReadyPrepTM 2-D cleanup kit (BioRad, Germany), according to the protocol provided by the manufacturer. .. The dried precipitate was dissolved in 125 μl of 2-D sample solution ((7 M urea, 2 M thiourea, 4%CHAPS, 40 mM DTT, 0.2%(w/v) ampholytes (pH 3–10)) and applied onto IPG strips (7 cm, pH 3–10 NL R, BioRad, Germany) for rehydration (12 h in RT).
Article Title: Interaction of serotonin transporter and TGFB1 facilitates histone serotonylation-mediated synaptic plasticity following environmental enrichment in stressed mouse Mus booduga.
Article Snippet: The membrane was then documented and the intensity of specific bands was measured using ChemiDoc XRS Image Lab-2 (Bio-Rad Laboratories, Inc., USA). .. 2-Dimensional gel based western blotting analysis The purified protein sample (200μg; Cat # 1632130, 2-D cleanup kit- Bio-Rad Laboratories, Inc., USA) was passively rehydrated on IPG Strips (pH: 3-10, 7 cm, ReadyStripTM IPG strip, Bio-Rad Laboratories, Inc.) for 16 h. Then, IPG strips were subjected to isoelectric focusing (IEF; Bio-Rad PROTEAN® IEF cell) in four steps: i) a rapid phase – 500 V (15 min), ii) gradual phase – 4000 V (1 h), iii) rapid phase – 4000 V (15000 V/h), iv) a hold step at 500 V. After completion of IEF, the strips were reduced and alkylated using equilibration buffers I and II (10 min/each) (ReadyPrepTM 2-D starter kit). .. Proteins from the IPG strip were separated in the second dimension (10% TGX stain free acrylamide kit, Cat# 1610183, BioRad Laboratories, Inc.) and subjected to western blot analysis using anti-SERT rabbit polyclonal antibody (Cat # AB9726, 1:2000; Merck Millipore), anti- TGFB1rabbit polyclonal antibody (Cat # 3711, 1:1000; Cell Signaling Technology), and secondary antibody goat antirabbit IgG ALP (Cat# 621100180011730; 1:5000; GeNei TM ), as mentioned earlier.
Stripping Membranes:Article Title: Interaction of serotonin transporter and TGFB1 facilitates histone serotonylation-mediated synaptic plasticity following environmental enrichment in stressed mouse Mus booduga
Article Snippet: The membrane was then documented and the intensity of specific bands was measured using ChemiDoc XRS Image Lab-2 (Bio-Rad Laboratories, Inc., USA). .. The purified protein sample (200 μg; Cat # 1632130, 2-D cleanup kit- Bio-Rad Laboratories, Inc., USA) was passively rehydrated on IPG Strips (pH: 3–10, 7 cm, ReadyStripTM IPG strip, Bio-Rad Laboratories, Inc.) for 16 h. Then, IPG strips were subjected to isoelectric focusing (IEF; Bio-Rad PROTEAN ® IEF cell) in four steps: (i) a rapid phase – 500 V (15 min), (ii) gradual phase – 4000 V (1 h), (iii) rapid phase – 4000 V (15000 V/h), (iv) a hold step at 500 V. After completion of IEF, the strips were reduced and alkylated using equilibration buffers I and II (10 min/each) (ReadyPrepTM 2-D starter kit). .. Proteins from the IPG strip were separated in the second dimension (10% TGX stain free acrylamide kit, Cat# 1610183, Bio-Rad Laboratories, Inc.) and subjected to western blot analysis using anti-SERT rabbit polyclonal antibody (Cat # AB9726, 1:2000; Merck Millipore), anti- TGFB1rabbit polyclonal antibody (Cat # 3711, 1:1000; Cell Signaling Technology), and secondary antibody goat anti-rabbit IgG ALP (Cat# 621100180011730; 1:5000; GeNeiTM), as mentioned earlier.
Article Title: Interaction of serotonin transporter and TGFB1 facilitates histone serotonylation-mediated synaptic plasticity following environmental enrichment in stressed mouse Mus booduga.
Article Snippet: The membrane was then documented and the intensity of specific bands was measured using ChemiDoc XRS Image Lab-2 (Bio-Rad Laboratories, Inc., USA). .. 2-Dimensional gel based western blotting analysis The purified protein sample (200μg; Cat # 1632130, 2-D cleanup kit- Bio-Rad Laboratories, Inc., USA) was passively rehydrated on IPG Strips (pH: 3-10, 7 cm, ReadyStripTM IPG strip, Bio-Rad Laboratories, Inc.) for 16 h. Then, IPG strips were subjected to isoelectric focusing (IEF; Bio-Rad PROTEAN® IEF cell) in four steps: i) a rapid phase – 500 V (15 min), ii) gradual phase – 4000 V (1 h), iii) rapid phase – 4000 V (15000 V/h), iv) a hold step at 500 V. After completion of IEF, the strips were reduced and alkylated using equilibration buffers I and II (10 min/each) (ReadyPrepTM 2-D starter kit). .. Proteins from the IPG strip were separated in the second dimension (10% TGX stain free acrylamide kit, Cat# 1610183, BioRad Laboratories, Inc.) and subjected to western blot analysis using anti-SERT rabbit polyclonal antibody (Cat # AB9726, 1:2000; Merck Millipore), anti- TGFB1rabbit polyclonal antibody (Cat # 3711, 1:1000; Cell Signaling Technology), and secondary antibody goat antirabbit IgG ALP (Cat# 621100180011730; 1:5000; GeNei TM ), as mentioned earlier.
Electrofocusing:Article Title: Interaction of serotonin transporter and TGFB1 facilitates histone serotonylation-mediated synaptic plasticity following environmental enrichment in stressed mouse Mus booduga
Article Snippet: The membrane was then documented and the intensity of specific bands was measured using ChemiDoc XRS Image Lab-2 (Bio-Rad Laboratories, Inc., USA). .. The purified protein sample (200 μg; Cat # 1632130, 2-D cleanup kit- Bio-Rad Laboratories, Inc., USA) was passively rehydrated on IPG Strips (pH: 3–10, 7 cm, ReadyStripTM IPG strip, Bio-Rad Laboratories, Inc.) for 16 h. Then, IPG strips were subjected to isoelectric focusing (IEF; Bio-Rad PROTEAN ® IEF cell) in four steps: (i) a rapid phase – 500 V (15 min), (ii) gradual phase – 4000 V (1 h), (iii) rapid phase – 4000 V (15000 V/h), (iv) a hold step at 500 V. After completion of IEF, the strips were reduced and alkylated using equilibration buffers I and II (10 min/each) (ReadyPrepTM 2-D starter kit). .. Proteins from the IPG strip were separated in the second dimension (10% TGX stain free acrylamide kit, Cat# 1610183, Bio-Rad Laboratories, Inc.) and subjected to western blot analysis using anti-SERT rabbit polyclonal antibody (Cat # AB9726, 1:2000; Merck Millipore), anti- TGFB1rabbit polyclonal antibody (Cat # 3711, 1:1000; Cell Signaling Technology), and secondary antibody goat anti-rabbit IgG ALP (Cat# 621100180011730; 1:5000; GeNeiTM), as mentioned earlier.
Article Title: Interaction of serotonin transporter and TGFB1 facilitates histone serotonylation-mediated synaptic plasticity following environmental enrichment in stressed mouse Mus booduga.
Article Snippet: The membrane was then documented and the intensity of specific bands was measured using ChemiDoc XRS Image Lab-2 (Bio-Rad Laboratories, Inc., USA). .. 2-Dimensional gel based western blotting analysis The purified protein sample (200μg; Cat # 1632130, 2-D cleanup kit- Bio-Rad Laboratories, Inc., USA) was passively rehydrated on IPG Strips (pH: 3-10, 7 cm, ReadyStripTM IPG strip, Bio-Rad Laboratories, Inc.) for 16 h. Then, IPG strips were subjected to isoelectric focusing (IEF; Bio-Rad PROTEAN® IEF cell) in four steps: i) a rapid phase – 500 V (15 min), ii) gradual phase – 4000 V (1 h), iii) rapid phase – 4000 V (15000 V/h), iv) a hold step at 500 V. After completion of IEF, the strips were reduced and alkylated using equilibration buffers I and II (10 min/each) (ReadyPrepTM 2-D starter kit). .. Proteins from the IPG strip were separated in the second dimension (10% TGX stain free acrylamide kit, Cat# 1610183, BioRad Laboratories, Inc.) and subjected to western blot analysis using anti-SERT rabbit polyclonal antibody (Cat # AB9726, 1:2000; Merck Millipore), anti- TGFB1rabbit polyclonal antibody (Cat # 3711, 1:1000; Cell Signaling Technology), and secondary antibody goat antirabbit IgG ALP (Cat# 621100180011730; 1:5000; GeNei TM ), as mentioned earlier.
Two-Dimensional Gel Electrophoresis:Article Title: P1PK blood group antigens in birds: implications for veterinary blood transfusion, human and pet health
Article Snippet: Lyophilized pigeons RBC membranes (60 mg) were suspended in 500 μl lysis buffer (250 mM Tris–HCl pH 8.0, 50 mM NaCl, and 1X proteinases inhibitor cocktail) (Thermo Fisher Scientific, USA), passed through a 22G × 1 1⁄2 needle 20 times, and centrifuged for 15 min with a maximum speed (15,000 × g) of 4 0 C. The supernatant (protein lysate) was collected for additional purification before isoelectric focusing. (IEF). .. The protein lysate (100 μl) was purified before the first dimension run of 2D electrophoresis using ReadyPrepTM 2-D cleanup kit (BioRad, Germany), according to the protocol provided by the manufacturer. .. The dried precipitate was dissolved in 125 μl of 2-D sample solution ((7 M urea, 2 M thiourea, 4%CHAPS, 40 mM DTT, 0.2%(w/v) ampholytes (pH 3–10)) and applied onto IPG strips (7 cm, pH 3–10 NL R, BioRad, Germany) for rehydration (12 h in RT).
Western Blot:Article Title: Interaction of serotonin transporter and TGFB1 facilitates histone serotonylation-mediated synaptic plasticity following environmental enrichment in stressed mouse Mus booduga.
Article Snippet: The membrane was then documented and the intensity of specific bands was measured using ChemiDoc XRS Image Lab-2 (Bio-Rad Laboratories, Inc., USA). .. 2-Dimensional gel based western blotting analysis The purified protein sample (200μg; Cat # 1632130, 2-D cleanup kit- Bio-Rad Laboratories, Inc., USA) was passively rehydrated on IPG Strips (pH: 3-10, 7 cm, ReadyStripTM IPG strip, Bio-Rad Laboratories, Inc.) for 16 h. Then, IPG strips were subjected to isoelectric focusing (IEF; Bio-Rad PROTEAN® IEF cell) in four steps: i) a rapid phase – 500 V (15 min), ii) gradual phase – 4000 V (1 h), iii) rapid phase – 4000 V (15000 V/h), iv) a hold step at 500 V. After completion of IEF, the strips were reduced and alkylated using equilibration buffers I and II (10 min/each) (ReadyPrepTM 2-D starter kit). .. Proteins from the IPG strip were separated in the second dimension (10% TGX stain free acrylamide kit, Cat# 1610183, BioRad Laboratories, Inc.) and subjected to western blot analysis using anti-SERT rabbit polyclonal antibody (Cat # AB9726, 1:2000; Merck Millipore), anti- TGFB1rabbit polyclonal antibody (Cat # 3711, 1:1000; Cell Signaling Technology), and secondary antibody goat antirabbit IgG ALP (Cat# 621100180011730; 1:5000; GeNei TM ), as mentioned earlier.
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