Primer3 was a complete re-implementation of an earlier program: Primer 0.5 (Steve Lincoln, Mark Daly, and Eric S. Lander). 18S levels were used for normalization. Furthermore, 45 g total protein of a mouse liver lysate 71 was added. Primer3 web service at primer3.ut.ee is a part of services provided by ELIXIR - European research infrastructure for biological information. Total Protein NormalizationTPN Next, 0.5 L of 1 N NaOH was added to maintain a basic pH. For the preparing 500 ml of ponceau S staining solution add 25 ml of glacial acetic acid to 400 ml of distilled water. In: Posch A. Proteinprotein interactions in cells are rapidly identified with improved proximity labeling methods. Add 0.5 gm of ponceau S tetrasodium salt to the acetic acid prepared above. MNase-digested samples were separated on 1.5% agarose gels and mononucleosome bands were excised. The total density for each lane is measured from the blot and a lane profile is obtained. Total RNA from the liver was extracted with Trizol reagent (Thermo Scientific) and reverse transcribed into cDNA using the High-Capacity cDNA kit (Applied Biosystems, Waltham, Massachusetts, USA). Glass bead lysis was performed at 4C four times 90 sec with 2 min on ice in between. Cold acetone was used to wash the protein precipitates twice. 1 mg of total protein was digested in RB (10 mM Tris-HCl pH 8, 2 mM CaCl 2 and 20 units Micrococcal nuclease (NEB)) in a total reaction volume of 200 l. Per cell lysate sample, 45 g total protein was loaded. Moritz, C. P. Tubulin or Not Tubulin: Heading Toward Total Protein Staining as Loading Control in Western Blots. MNase-digested samples were separated on 1.5% agarose gels and mononucleosome bands were excised. Preparation of ponceau S staining solution. Ponceau S, loading control. Protein levels were normalized by total protein loading (Ponceau staining). Then, protein precipitates were resuspended in 20 L of 10 mM Tris-HCl pH = 8, followed by the addition of 20 L of (2) Tris-Glycine SDS Sample Buffer supplemented with 5% (v/v) -mercaptoethanol. 27 Stain-Free Imaging Technology. Next, 0.5 L of 1 N NaOH was added to maintain a basic pH. The samples to be analyzed were mixed with an appropriate volume of 5 or 2 SDS sample buffer and denatured for 10 min at 95 C. Proteinprotein interactions in cells are rapidly identified with improved proximity labeling methods. (2015) Western Blotting Using In-Gel Protein Labeling as a Normalization Control: Stain-Free Technology. 3. Total RNA from the liver was extracted with Trizol reagent (Thermo Scientific) and reverse transcribed into cDNA using the High-Capacity cDNA kit (Applied Biosystems, Waltham, Massachusetts, USA). Make up the solution to 500 ml using distilled water. (eds) Proteomic Profiling. Stain-Free total protein normalization is performed by measuring total protein directly on the membrane that is used for western blotting. C, Comparison of RBM22 protein levels from PIN (n = 5), MD-PCa (n = 4) and PD-PCa (n = 5) derived from TRAMP mice. 1 mg of total protein was digested in RB (10 mM Tris-HCl pH 8, 2 mM CaCl 2 and 20 units Micrococcal nuclease (NEB)) in a total reaction volume of 200 l. Here, instead of the arithmetic mean and s.d. Stain-Free Imaging Technology. Proteomics 17, (2017). Add a thin layer of PBS (~750 L for a 6 cm dish) for both a "mock" treated and for an Ionizing Radiation (IR) treated condition. Repeat this step for a total of two washes. Ponceau S, loading control. Advantages of Total Protein Normalization. 2. Experiments using the PathScan Intracellular Signaling Array Kit (Chemiluminescent Readout, #7323) from Cell Signaling were done according to the manufacturers instructions. In: Posch A. 2. 27 Wash the Ponceau off with water. Primer3 web service at primer3.ut.ee is a part of services provided by ELIXIR - European research infrastructure for biological information. Then, protein precipitates were resuspended in 20 L of 10 mM Tris-HCl pH = 8, followed by the addition of 20 L of (2) Tris-Glycine SDS Sample Buffer supplemented with 5% (v/v) -mercaptoethanol. Next, 0.5 L of 1 N NaOH was added to maintain a basic pH. Here, instead of the arithmetic mean and s.d. Methods in Molecular Biology, vol 1295. Total Protein Normalization. Gilda J.E., Gomes A.V. (eds) Proteomic Profiling. Stain with Ponceau S solution to verify the quality of the transfer and that protein loading is comparable between samples. Proteomics 17, (2017). Protein levels were normalized by total protein loading (Ponceau staining). Advantages of Total Protein Normalization. Glass bead lysis was performed at 4C four times 90 sec with 2 min on ice in between. Methods in Molecular Biology, vol 1295. Protein loading into SDS-PAGE was normalized by staining the nitrocellulose membranes with Ponceau S. 2.5. The body weight of O-CR rats was reduced by an average of one-third (32.6% 3.9%) of that of O-AL control rats after 9 months of CR, with no effect on serum glucose levels (Figure 1B).CR extended both the median and maximum lifespans of O-CR rats compared with those of O-AL control rats (Figure 1B).In addition, CR exerted profound effects on aging-associated Then, 1530 g of total protein was loaded per sample and electrophoresis was performed in 1 Tris-glycine SDS running buffer (25 mM Tris, 250 mM glycine, 0.1% [w/v] SDS) with 30 mA/gel. 1 mg of total protein was digested in RB (10 mM Tris-HCl pH 8, 2 mM CaCl 2 and 20 units Micrococcal nuclease (NEB)) in a total reaction volume of 200 l. The body weight of O-CR rats was reduced by an average of one-third (32.6% 3.9%) of that of O-AL control rats after 9 months of CR, with no effect on serum glucose levels (Figure 1B).CR extended both the median and maximum lifespans of O-CR rats compared with those of O-AL control rats (Figure 1B).In addition, CR exerted profound effects on aging-associated Asterisks (** P < 0.01) indicate statistically significant differences between groups. Stir the mixture to dissolve. Total RNA from the liver was extracted with Trizol reagent (Thermo Scientific) and reverse transcribed into cDNA using the High-Capacity cDNA kit (Applied Biosystems, Waltham, Massachusetts, USA). The body weight of O-CR rats was reduced by an average of one-third (32.6% 3.9%) of that of O-AL control rats after 9 months of CR, with no effect on serum glucose levels (Figure 1B).CR extended both the median and maximum lifespans of O-CR rats compared with those of O-AL control rats (Figure 1B).In addition, CR exerted profound effects on aging-associated The samples to be analyzed were mixed with an appropriate volume of 5 or 2 SDS sample buffer and denatured for 10 min at 95 C. The use of total protein measurement for western blot loading controls (total protein normalization, TPN) is a method devised to minimize the inherent difficulties with using housekeeping proteins. Advantages of Total Protein Normalization. Membranes were then stained with Ponceau S to confirm the quality of the transfer and equal loading of samples. G protein-coupled receptors (GPCRs) regulate processes ranging from immune responses to neuronal signaling. The samples to be analyzed were mixed with an appropriate volume of 5 or 2 SDS sample buffer and denatured for 10 min at 95 C. Membranes were then stained with Ponceau S to confirm the quality of the transfer and equal loading of samples. Healthcare). Total gDNA was then extracted using the Gentra Puregen Blood kit (QIAGEN) according to the manufacturers instructions. Asterisks (** P < 0.01) indicate statistically significant differences between groups. 27 C, Comparison of RBM22 protein levels from PIN (n = 5), MD-PCa (n = 4) and PD-PCa (n = 5) derived from TRAMP mice. (2015) Western Blotting Using In-Gel Protein Labeling as a Normalization Control: Stain-Free Technology. The total density for each lane is measured from the blot and a lane profile is obtained. Data are mean s.e.m., n = 8 biologically independent leaf discs. Gilda J.E., Gomes A.V. Add 0.5 gm of ponceau S tetrasodium salt to the acetic acid prepared above. Wash the Ponceau off with water. Add 0.5 gm of ponceau S tetrasodium salt to the acetic acid prepared above. Total Protein Normalization. Proteomics 17, (2017). Cold acetone was used to wash the protein precipitates twice. Protocols for AAV barcoding and NGS analysis as well as the associated in-house normalization strategy have all been Qiagen) by the FastPrep-24 (MP Biomedicals) device for 40 s at the speed of 5 m/s. Healthcare). Abdominal fat has been shown to increase cardiometabolic disease risk. Preparation of ponceau S staining solution. Total Protein NormalizationTPN Membranes were then stained with Ponceau S to confirm the quality of the transfer and equal loading of samples. Healthcare). Ponceau-S (H 2 O 950 ml/glacial acetic acid 50 ml/Ponceau Red dye 1 g) staining was performed after incubation of nitrocellulose membrane for 5 min. In order to avoid noise of signal, total protein staining should be performed before blocking of the membrane. Total protein levels can be determined by staining the membrane with total protein stains (e.g. Here, instead of the arithmetic mean and s.d. Abdominal fat has been shown to increase cardiometabolic disease risk. Total Protein NormalizationTPN Furthermore, 45 g total protein of a mouse liver lysate 71 was added. The total density for each lane is measured from the blot and a lane profile is obtained. Total gDNA was then extracted using the Gentra Puregen Blood kit (QIAGEN) according to the manufacturers instructions. Use a Ponceau stain to monitor for the equal loading of samples. Destain with deionized water and rinse once with 1 TBST. Then, protein precipitates were resuspended in 20 L of 10 mM Tris-HCl pH = 8, followed by the addition of 20 L of (2) Tris-Glycine SDS Sample Buffer supplemented with 5% (v/v) -mercaptoethanol. across all clusters, only clusters with probabilities between the 5th and 95th percentiles were used to calculate the mean and s.d. Ponceau-S (H 2 O 950 ml/glacial acetic acid 50 ml/Ponceau Red dye 1 g) staining was performed after incubation of nitrocellulose membrane for 5 min. Equal amounts of protein were loaded and separated on Criterion XT Bis-Tris Gels (Bio-Rad, Hercules, CA, USA) and transferred to polyvinylidene fluoride membranes (Merck). At least seven different approaches for total protein staining have been described for western blot normalization: Ponceau S, stain-free techniques, Sypro Ruby, Epicocconone, Coomassie R-350, Amido Black, and Cy5. Stain-Free total protein normalization is performed by measuring total protein directly on the membrane that is used for western blotting. Proteinprotein interactions in cells are rapidly identified with improved proximity labeling methods. Glass bead lysis was performed at 4C four times 90 sec with 2 min on ice in between. Then, 1530 g of total protein was loaded per sample and electrophoresis was performed in 1 Tris-glycine SDS running buffer (25 mM Tris, 250 mM glycine, 0.1% [w/v] SDS) with 30 mA/gel. Total Protein Normalization. Wash the Ponceau off with water. Expression profiles are represented as read counts, and normalization values are based on the transcript length and coverage depth. Repeat this step for a total of two washes. Make up the solution to 500 ml using distilled water. Stain with Ponceau S solution to verify the quality of the transfer and that protein loading is comparable between samples. Ponceau-S (H 2 O 950 ml/glacial acetic acid 50 ml/Ponceau Red dye 1 g) staining was performed after incubation of nitrocellulose membrane for 5 min. Total gDNA was then extracted using the Gentra Puregen Blood kit (QIAGEN) according to the manufacturers instructions. Stain with Ponceau S solution to verify the quality of the transfer and that protein loading is comparable between samples. MNase-digested samples were separated on 1.5% agarose gels and mononucleosome bands were excised. Methods in Molecular Biology, vol 1295. across all clusters, only clusters with probabilities between the 5th and 95th percentiles were used to calculate the mean and s.d. Expression profiles are represented as read counts, and normalization values are based on the transcript length and coverage depth. G protein-coupled receptors (GPCRs) regulate processes ranging from immune responses to neuronal signaling. In order to avoid noise of signal, total protein staining should be performed before blocking of the membrane. Gilda J.E., Gomes A.V. 2. Stir the mixture to dissolve. Total protein levels can be determined by staining the membrane with total protein stains (e.g. Primer3 was a complete re-implementation of an earlier program: Primer 0.5 (Steve Lincoln, Mark Daly, and Eric S. Lander). The kinetics of ROS production (e) and total ROS production (f) are shown, as mean values of total photon counts over 32 min. In order to avoid noise of signal, total protein staining should be performed before blocking of the membrane. The use of total protein measurement for western blot loading controls (total protein normalization, TPN) is a method devised to minimize the inherent difficulties with using housekeeping proteins. Moritz, C. P. Tubulin or Not Tubulin: Heading Toward Total Protein Staining as Loading Control in Western Blots. Total protein levels can be determined by staining the membrane with total protein stains (e.g. C, Comparison of RBM22 protein levels from PIN (n = 5), MD-PCa (n = 4) and PD-PCa (n = 5) derived from TRAMP mice. Primer3 web service at primer3.ut.ee is a part of services provided by ELIXIR - European research infrastructure for biological information. Protein levels were normalized by total protein loading (Ponceau staining). Real-time polymerase chain reaction. Stain-Free total protein normalization is performed by measuring total protein directly on the membrane that is used for western blotting. Preparation of ponceau S staining solution. Ponceau S, loading control. Moritz, C. P. Tubulin or Not Tubulin: Heading Toward Total Protein Staining as Loading Control in Western Blots. Add a thin layer of PBS (~750 L for a 6 cm dish) for both a "mock" treated and for an Ionizing Radiation (IR) treated condition. (2015) Western Blotting Using In-Gel Protein Labeling as a Normalization Control: Stain-Free Technology. The kinetics of ROS production (e) and total ROS production (f) are shown, as mean values of total photon counts over 32 min. For the preparing 500 ml of ponceau S staining solution add 25 ml of glacial acetic acid to 400 ml of distilled water. Per cell lysate sample, 45 g total protein was loaded. Data are mean s.e.m., n = 8 biologically independent leaf discs. Protocols for AAV barcoding and NGS analysis as well as the associated in-house normalization strategy have all been Qiagen) by the FastPrep-24 (MP Biomedicals) device for 40 s at the speed of 5 m/s. 3. Add a thin layer of PBS (~750 L for a 6 cm dish) for both a "mock" treated and for an Ionizing Radiation (IR) treated condition. Asterisks (** P < 0.01) indicate statistically significant differences between groups. (eds) Proteomic Profiling. Real-time polymerase chain reaction. Primer3 was a complete re-implementation of an earlier program: Primer 0.5 (Steve Lincoln, Mark Daly, and Eric S. Lander). Real-time polymerase chain reaction. Make up the solution to 500 ml using distilled water. Destain with deionized water and rinse once with 1 TBST. Then, 1530 g of total protein was loaded per sample and electrophoresis was performed in 1 Tris-glycine SDS running buffer (25 mM Tris, 250 mM glycine, 0.1% [w/v] SDS) with 30 mA/gel. Experiments using the PathScan Intracellular Signaling Array Kit (Chemiluminescent Readout, #7323) from Cell Signaling were done according to the manufacturers instructions. Per cell lysate sample, 45 g total protein was loaded. At least seven different approaches for total protein staining have been described for western blot normalization: Ponceau S, stain-free techniques, Sypro Ruby, Epicocconone, Coomassie R-350, Amido Black, and Cy5. Furthermore, 45 g total protein of a mouse liver lysate 71 was added. At least seven different approaches for total protein staining have been described for western blot normalization: Ponceau S, stain-free techniques, Sypro Ruby, Epicocconone, Coomassie R-350, Amido Black, and Cy5. Stir the mixture to dissolve. Use a Ponceau stain to monitor for the equal loading of samples. The kinetics of ROS production (e) and total ROS production (f) are shown, as mean values of total photon counts over 32 min. Equal amounts of protein were loaded and separated on Criterion XT Bis-Tris Gels (Bio-Rad, Hercules, CA, USA) and transferred to polyvinylidene fluoride membranes (Merck). Protocols for AAV barcoding and NGS analysis as well as the associated in-house normalization strategy have all been Qiagen) by the FastPrep-24 (MP Biomedicals) device for 40 s at the speed of 5 m/s. Stain-Free Imaging Technology. Protein loading into SDS-PAGE was normalized by staining the nitrocellulose membranes with Ponceau S. 2.5. Abdominal fat has been shown to increase cardiometabolic disease risk. Destain with deionized water and rinse once with 1 TBST. Experiments using the PathScan Intracellular Signaling Array Kit (Chemiluminescent Readout, #7323) from Cell Signaling were done according to the manufacturers instructions. The use of total protein measurement for western blot loading controls (total protein normalization, TPN) is a method devised to minimize the inherent difficulties with using housekeeping proteins. For the preparing 500 ml of ponceau S staining solution add 25 ml of glacial acetic acid to 400 ml of distilled water. 3. Protein loading into SDS-PAGE was normalized by staining the nitrocellulose membranes with Ponceau S. 2.5. Equal amounts of protein were loaded and separated on Criterion XT Bis-Tris Gels (Bio-Rad, Hercules, CA, USA) and transferred to polyvinylidene fluoride membranes (Merck). Repeat this step for a total of two washes. 18S levels were used for normalization. 18S levels were used for normalization. Cold acetone was used to wash the protein precipitates twice. across all clusters, only clusters with probabilities between the 5th and 95th percentiles were used to calculate the mean and s.d. Data are mean s.e.m., n = 8 biologically independent leaf discs. In: Posch A. 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