Anti-PON1, PON2, and PON3 antibodies were from Abcam (Cambridge, MA, USA). Anti β-actin antibody was from Sigma-Aldrich (St. Louis, MO, USA), while 10× Tris-buffered saline, Tween-20, Coomassie Brilliant Blue R-250 were from Bio-Rad Laboratories (Hercules, CA, USA). XCell II™ Blot Module, XCell SureLock™ Electrophoresis Cell, NuPAGE® MOPS SDS Running Buffer 20×, NuPAGE® LDS Sample Buffer 4×, NuPAGE® Antioxidant, NuPAGE® Sample Reducing Agent 10× and NuPAGE® 10% Bis-Tris Protein Gels were from Life Technologies (Carlsbad, CA, USA). Immobilon®-P Transfer Membrane was from Millipore Corporation (Billerica, MA, USA). Restore™ Western Blot Stripping Buffer, PageRuler™ Prestained Protein Ladder, SuperSignal® West Pico Chemiluminescent Substrate, SuperScript® III Reverse Transcriptase and TaqMan® Gene Expression Assays were from Thermo-Fisher Scientific (Waltham, MA, USA). Antirabbit IgG HRP-linked antibody and Cell Lysis Buffer 10× were from Cell Signaling Technology (Danvers, MA, USA). HRP Goat Anti-Mouse Ig was from BD Biosciences (San Jose, CA, USA). RNeasy® Mini Kit was purchased from Qiagen (Hilden, Germany). TaqMan® Gene Expression Master Mix was from Applied Biosystems (Foster City, CA, USA).
Nupage lds sample buffer 4
Manufactured by Thermo Fisher Scientific
Sourced in United States
NuPAGE LDS Sample Buffer (4×) is a ready-to-use buffer used to prepare protein samples for electrophoresis analysis. It is designed to denature and solubilize proteins prior to gel separation.
Automatically generated - may contain errors
Lab products found in correlation
Nupage sample reducing agent 10, by Thermo Fisher Scientific (9 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (6 mentions)
Pageruler prestained protein ladder, by Thermo Fisher Scientific (4 mentions)
Bis tris gel, by Thermo Fisher Scientific (4 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (4 mentions)
Nupage 4 12 bis tris protein gel, by Thermo Fisher Scientific (4 mentions)
Odyssey blocking buffer, by LI COR (3 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Chemidoc mp imaging system, by Bio-Rad (3 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (3 mentions)
Anti β actin antibody, by Merck Group (2 mentions)
Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Protein sample loading buffer, by LI COR (2 mentions)
Pampkα1 α2, by Cell Signaling Technology (2 mentions)
75 protocols using nupage lds sample buffer 4
1
Western Blot Analysis of PON Proteins
2
SDS-PAGE and Western Blotting for Protein Analysis
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Sodium dodecyl sulfate–polyacrylamide
gel electrophoresis (SDS–PAGE) was used to separate protein
samples prior to Western blotting. The protein sample (20 μL)
was prepared in NuPAGE LDS Sample Buffer (4×) (LifeTechnologies)
and NuPAGE Sample Reducing Agent (10×) (Life Technologies) and
run on a NuPAGE 4 to 12% gradient Bis-Tris (Life Technologies) gel
with MES running buffer (Life Technologies) (200 V, 35 min). The proteins
were transferred from the gel to an iBlot Transfer Stack with a polyvinylidene
fluoride (PVDF) membrane (0.2 μm pore size) using the iBlot
Dry Blotting System (Life Technologies) according to the manufacturer’s
protocol. After the transfer, the PVDF membrane was blocked with 5%
(w/v) dry skimmed milk powder in PBS with 0.05% Tween 20 (blocking
buffer) (RT, 1 h, gentle agitation). The membrane was then incubated
with primary antibodies in blocking buffer (1:1000, 4 °C overnight
or RT for 1 h) followed by washing four times with PBS-T (RT, 10 min).
After being washed, the membrane was incubated with Alexa Fluor 488-
or 594-labeled secondary antibody (1:1000, RT, 1 h). After incubation,
the membrane was washed four times with PBS-T and imaged using a Typhoon
9400 laser-based scanner (GE Healthcare) at 550 V using a green (532
nm) excitation laser to excite Alexa Fluor 594 or a blue (488 nm)
laser to excite Alexa Fluor 488.
gel electrophoresis (SDS–PAGE) was used to separate protein
samples prior to Western blotting. The protein sample (20 μL)
was prepared in NuPAGE LDS Sample Buffer (4×) (LifeTechnologies)
and NuPAGE Sample Reducing Agent (10×) (Life Technologies) and
run on a NuPAGE 4 to 12% gradient Bis-Tris (Life Technologies) gel
with MES running buffer (Life Technologies) (200 V, 35 min). The proteins
were transferred from the gel to an iBlot Transfer Stack with a polyvinylidene
fluoride (PVDF) membrane (0.2 μm pore size) using the iBlot
Dry Blotting System (Life Technologies) according to the manufacturer’s
protocol. After the transfer, the PVDF membrane was blocked with 5%
(w/v) dry skimmed milk powder in PBS with 0.05% Tween 20 (blocking
buffer) (RT, 1 h, gentle agitation). The membrane was then incubated
with primary antibodies in blocking buffer (1:1000, 4 °C overnight
or RT for 1 h) followed by washing four times with PBS-T (RT, 10 min).
After being washed, the membrane was incubated with Alexa Fluor 488-
or 594-labeled secondary antibody (1:1000, RT, 1 h). After incubation,
the membrane was washed four times with PBS-T and imaged using a Typhoon
9400 laser-based scanner (GE Healthcare) at 550 V using a green (532
nm) excitation laser to excite Alexa Fluor 594 or a blue (488 nm)
laser to excite Alexa Fluor 488.
3
Western Blot Analysis of BMP7 Signaling
4
Quantification of AMPK Phosphorylation
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Quantitation of AMPK phosphorylation proteins was carried out by western blot analysis. The white adipose tissues of each rat were homogenized in a CelLytic™ MT lysis reagent (Sigma–Aldrich, Sigma, St. Louis, MO, USA) and centrifuged at 12,000g for 20 min at 4°C. The protein content of the clear lysates was estimated with the Bradford method using the Protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA). Forty micrograms of protein were dissolved in NuPAGE® LDS sample buffer 4× (Life Technologies, Gaithersburg, MD, USA). Membranes were incubated for 1 h in a blocking solution containing 5% non-fat milk in Tris-buffered saline and then incubated for 12 h at 4°C with anti-β-actin (1:1,000, Abcam, Cambridge, England) and anti-phospho-AMPK (1:1,000, Abcam). After incubation with the primary antibody, membranes were incubated with a secondary antibody (anti-rabbit IgG HRP-linked antibody, 1:5,000, Cell Signaling, Beverly, MA, USA) for 1 h at room temperature. Proteins were developed using a detection system (Molecular Imager ChemiDoc™XRS+, Bio-Rad Laboratories) and visualized with Image Lab™ Software (Bio-Rad Laboratories).
5
Western Blot Analysis of hTERT Expression
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Protein samples were mixed with one-third volume of NuPAGE® LDS Sample Buffer (4×) (Life Technologies), boiled at 95°C for 5 min, and then electrophoresed on a 4–12% Bis-Tris gel (Life Technologies). Standard SDS-PAGE and western blotting protocols were carried out afterwards. Primary antibodies used were as follows: anti-hTERT antibody (Abcam, ab32020, 1:1000), anti-β-actin antibody (Sigma, A5441, 1:5000). Secondary antibodies used were as follows: peroxidase-AffiniPure donkey anti-rabbit IgG (H + L) (Jackson, 711-035-152, 1:5000), peroxidase-AffiniPure donkey anti-mouse IgG (H + L) (Jackson, 715-035-150, 1:5000). SuperSignal® West Pico Chemiluminescent Substrate (Thermo Scientific) was used to generate signals on western blots. The signals were detected with a FluorChem HD2 imaging system (Alpha Innotech) and quantified with ImageQuant TL v2005 software.
6
Metformin Regulation of AMPK Signaling
7
AICAR Signaling Pathway Analysis
8
Recombinant Protein Analysis by SDS-PAGE
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One milliliter of the culture was centrifuged, and the supernatant was collected every 6 h. Next, 12% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the recombinant protein. The samples were mixed with NuPAGE LDS sample buffer (4 ×) (Life Technology, Carlsbad, CA, USA) and heated at 70 °C for 10 min before loading on the gel. The electrophoresis was performed at a constant voltage of 200 V for 35 min, and the gel was stained with Coomassie brilliant blue R-250.
9
Western Blot Quantification Protocol
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Treated tissues or cells were washed with precooled PBS and then lysed with an appropriate volume of RIPA lysis buffer (P0013C, Beyotime, Haimen, China) containing protease inhibitors; following centrifugation, the supernatant was collected. The total protein concentration of each sample was determined by the bicinchoninic acid (BCA) method (Solarbio, Beijing, China). Protein samples were boiled in NuPAGE™ LDS sample buffer (4×) (#84788, Thermo Fisher Scientific, Waltham, MA, USA) for denaturation and stability. Then, 10% SDS-PAGE gels were prepared, and 40 μg of protein samples were added. After the markers bands had completely separated by constant pressure electrophoresis, the proteins were transferred to a 0.22-μm polyvinylidene difluoride (PVDF) membrane at constant pressure for approximately 90 min. Membranes were blocked with 5% skimmed milk at room temperature for 1 h, followed by incubation with primary and secondary antibodies, enhanced chemiluminescent (ECL) development, and imaging. Finally, the gray values of immunoreactive bands for each target protein were analyzed with Image J software. Statistical analyses were performed using Graph Prism 8 (GraphPad Software, San Diego, CA, USA). Information on all antibodies used for Western blotting are listed in Table 1 .
10
Western Blot Analysis of Antigen-Stimulated Mast Cells
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MCs were stimulated with antigen for 15 min with or without dasatinib or PP2 and then placed on ice. The cells were washed 3 times with ice-cold PBS and the cells were lysed using the RIPA buffer (Thermo Fisher scientific, Waltham, MA, USA) in 1 mM phenylmethylsulfonyl fluoride, 2.5 mM p-nitrophenyl phosphate, 0.7 μg/mL pepstatin, and the protease-inhibitor cocktail (Sigma-Aldrich). The cell lysates were centrifuged at 15,000×g for 5 min and the equal amount of protein from the lysates was denatured at 100°C for 5 min with a NuPAGE™LDS sample buffer (4×) (Thermo Fisher Scientific). Proteins were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Berkeley, CA, USA). The membranes were blocked in 5% BSA for 1 h and then incubated overnight with each primary antibody in the TBS-T buffer (tris-buffered saline with 0.1% tween 20) contained 5% BSA at 4°C. The membranes were washed 3 times with TBS-T buffer and incubated in a horse-radish peroxidase-coupled secondary antibody at room temperature for 1 h. The membranes were washed, and treated with chemiluminescence reagents (Thermo Fisher scientific) according to the manufacturer’s guideline and then visualized and quantified by the ImageQuantTMLAS 4000 system (GE Healthcare Life Sciences, Piscataway, NJ, USA).
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