Cell wall extracts were prepared as described earlier (75 (link)) and quantified using a Qubit 2.0 fluorometer (Thermo Fisher Scientific). Equal amounts of the cell wall extracts were boiled in NuPAGE lithium dodecyl sulfate (LDS) sample buffer (Thermo Fisher Scientific), separated by SDS-PAGE on a NuPAGE 4 to 12% bis-Tris protein gradient gel (Thermo Fisher Scientific), and transferred to a polyvinylidene difluoride (PVDF) membrane using an iBlot transfer pack (Thermo Fisher Scientific). The membrane was blocked in casein blocking buffer (Sigma-Aldrich) and incubated for 1 hour with rabbit primary Pil3B antibodies (1:1,000) and subsequently with horseradish peroxidase-conjugated goat anti-rabbit antibody (1:5,000). The membrane was washed 3 times with PBS and 0.1% Tween 20 between the incubation with antibodies. Chemiluminescence was detected on a ChemiDoc gel imaging system (Bio-Rad Laboratories).
Nu page lithium dodecyl sulfate sample buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The Nu-PAGE lithium dodecyl sulfate (LDS) sample buffer is a laboratory reagent used in the preparation of protein samples for electrophoresis analysis. It is designed to denature and solubilize proteins prior to separation on a polyacrylamide gel.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc mp imaging system, by Bio-Rad (6 mentions)
Nupage sample reducing agent, by Thermo Fisher Scientific (6 mentions)
Nitrocellulose membrane, by Bio-Rad (5 mentions)
Nupage 4 12 bis tris protein gel, by Thermo Fisher Scientific (3 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (3 mentions)
Complete mini, by Roche (3 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (3 mentions)
Image lab 5, by Bio-Rad (3 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions)
Nupage 4 to 12 bis tris protein gel, by Thermo Fisher Scientific (2 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions)
Nupage 4 12 bis tris polyacrylamide gel, by Thermo Fisher Scientific (2 mentions)
Coomassie brilliant blue g 250, by Merck Group (2 mentions)
Bis tris gel, by Thermo Fisher Scientific (2 mentions)
Xcell surelock mini cell electrophoresis system, by Thermo Fisher Scientific (2 mentions)
Tris acetate sds running buffer, by Thermo Fisher Scientific (2 mentions)
Pierce phosphatase inhibitor mini tablets, by Thermo Fisher Scientific (2 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (2 mentions)
Nupage 4 to 12 polyacrylamide bis tris gels, by Thermo Fisher Scientific (2 mentions)
50 protocols using nu page lithium dodecyl sulfate sample buffer
1
Cell Wall Protein Immunoblotting Protocol
2
Detailed Western Blotting Protocol
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Western blotting was performed as previously described (Lynch et al., 2021 (link)). Briefly, cells were counted, pelleted, resuspended in 1× NuPAGE lithium dodecyl sulfate (LDS) sample buffer (NP007; Thermo Fisher Scientific) + 5% 2-betamercaptoethanol at 300,000 cells per 200 µl, and boiled for 15 min. Protein lysates were separated by 13.5% SDS-PAGE gels using 1× NuPAGE MOPS buffer (NP0001; Thermo Fisher Scientific) at 75 V for 30 min, then 110 V for 100 min, and then wet transferred to a nitrocellulose membrane (Bio-Rad) at 100 V for 70 min using Transfer Buffer (25 mM Tris Base, 100 mM glycine, 20% methanol). Membranes were ponceau stained and imaged. Membranes were blocked in 5% milk in Tris-buffered saline with Tween (TBST) for 1 h and then probed with primary antibody overnight (see Table 2 ). Membranes were washed with TBST for 30 min, incubated with secondary antibodies conjugated to horseradish peroxidase (α-mouse or α-rabbit; 1:5,000) at room temperature for 1 h, washed with TBST for 30 min, and detected using Clarity Western ECL Substrate (1705061; Bio-Rad) and Chemidoc MP Imaging System (Bio-Rad). Images were formatted using Adobe Photoshop and Illustrator. Densitometry analysis was quantified using ImageJ.
3
SDS-PAGE Analysis of Purified FI Protein
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For analysis of the purified FI protein, samples were diluted 2x in Tris-buffered saline (TBS, 50mM trisHCl, 150mM NaCl, pH7.4), denatured with NuPAGE lithium dodecyl sulfate ((LDS) sample buffer (ThermoFisher Scientific, USA) under non-reducing conditions, and separated on NuPAGE 4-12% Bis-Tris gel (Invitrogen, USA). The NuPAGE were fixed (50% methanol, 12% acetic acid, 0.0185% formaldehyde), washed with 50% ethanol and sensitized (0.8 mM Na2S2O3·5H2O). After rinsing with water, the gels were stained (11.8 mM AgN03, 0.028% formaldehyde), rinsed again and then treated with developing solution (0.57M Na2CO3, 0.02 mM Na2S2O3·5H2O, 0.0185% formaldehyde). Developing was stopped by incubation with fixation solution prior to taking pictures.
4
Protein-Protein Interactions via Co-immunoprecipitation
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Co-immunoprecipitation (CoIP) was performed by using μMACS epitope tag protein isolation kits (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) according to manufacturer's instructions. Briefly, 24 h after transfection, HEK293 cells were lysed in 1 ml lysis buffer (Miltenyi Biotec) containing 1x proteinase and phosphatase inhibitor (HALT, Thermo Fisher). Cellular debris were pelleted and 1 ml of supernatants were incubated with anti-tag MicroBeads for 1 h on ice. Afterwards, cell lysates were applied onto equilibrated μmacs columns and washed stringently to remove non-specific interacting molecules. Proteins were eluted with 1x pre-heated 95°C NuPage lithium dodecyl sulfate (LDS) sample buffer (Thermo Fisher) supplemented with 100 mM Dithiothreitol (DTT, AppliChem, Darmstadt, Germany). Raw lysates and eluates were analyzed by Western blot. For endogenous CoIP, PHNs were lysed in lysis buffer (Miltenyi Biotec) supplemented with 1x HALT. Lysates were centrifuged and supernatant was transferred to fresh Eppendorf cups. Lysate of PHNs or human tissue was adjusted to 1 ml with lysis buffer. Afterwards, lysates were incubated with 2 μg of the required antibodies for 2 h at 4°C. Afterwards, cell lysates were incubated with anti protein G MicroBeads (Miltenyi Biotec) for 1 h on ice. Immunoprecipitations were performed as described above.
5
LRRK2 Immunoprecipitation and Phosphorylation
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Endogenous LRRK2 was immunoprecipitated as described above from lysates (3.5 mg of protein). Immunoprecipitated LRRK2 was washed twice with the lysis buffer containing 0.5 M NaCl and eluted from the beads with 30 μl of 2× NuPAGE lithium dodecyl sulfate (LDS) Sample Buffer (Thermo Fisher Scientific). Eluted samples at 5 and 15 μl were loaded for detecting total LRRK2 and phospho-Ser1292 LRRK2 respectively. For detecting phospho-Ser1292 LRRK2 VeriBlot secondary antibody (Abcam, ab131366) was used instead of normal anti-rabbit IgG secondary antibody.
6
Quantification of GSNOR Protein
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Seven-day-old seedlings (around 30 plants) grown on ½ MS medium or ½ MS medium supplemented with 300 µM Fe were harvested, weighed and ground in liquid nitrogen to fine powder. In total 5 volumes (5 µl. mg−1) of 2 × NuPAGE lithium dodecyl sulfate (LDS) sample buffer (Thermo-Fischer) with Tris(2-carboxyethyl) phosphine Hydrochloride (TCEP) was added to the samples and then boiled for 5 min. After centrifugation at 15,000 × g at 4 °C for 15 min, 15 μL of supernatant were loaded, separated in a NuPAGE 4–12% Bis-Tris protein gel (Thermo-Fischer), followed by a wet transfer to nitrocellulose membrane (Bio-Rad). The GSNOR protein was detected by probing the membrane with anti-GSNOR antibody (AS09 647, Agrisera) at a dilution of 1:2000. Loading was controlled by using a rabbit anti-histone H3 antibody (9715 S, Cell Signaling Technology) at a dilution of 1:2000. Signal was detected using the SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo-Fischer). At least two independent biological replicates (the samples from two different experiments) were performed for the western blot. The representative images from one experiment were presented.
7
Western Blot Analysis of Cell Lysates
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Cells were lysed with NuPage lithium dodecyl sulfate sample buffer (Thermo Fisher Scientific) at the concentration of 105 cells per 15 μl lithium dodecyl sulfate supplemented with 10% β-mercaptoethanol and benzonase nuclease (Sigma-Aldrich). The samples were then denatured at 70°C. Protein lysates were separated by SDS-PAGE on 4–12% Bis-Tris precase gels (Invitrogen) and transferred to a polyvinylidene difluoride membrane (Invitrogen) by iBlot (Thermo Fisher Scientific) or wet transfer. Membranes were then blocked in milk with 5% Tris-buffered saline with 0.01% Tween-20; TBST) for 1 h at room temperature and then incubated with primary antibody in milk or 5% BSA overnight at 4°C. The membrane was washed for 3 × 10 min with TBST at room temperature and then stained with HRP-linked secondary antibody in milk for 1 h at room temperature. After 3 × 10–min washes with TBST and 1 × 10–min wash with PBS, the membrane was exposed to enhanced chemiluminescent substrates (Thermo Fisher Scientific) and developed by film.
8
Aβ Protein Analysis in Mouse Brain
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On feeding day 58, the mouse brain cortex was homogenized with M-PER (Thermo Fisher Scientific, Waltham, MA, USA) containing 1 × Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Samples were mixed with NuPAGE lithium dodecyl sulfate sample buffer (Thermo Fisher Scientific) containing 5% 2-mercaptoethanol (Wako, Osaka, Japan) at 75 °C for 5 min and loaded onto a 14% sodium dodecyl sulfate polyacrylamide gel (SDS–PAGE) (50 µg/lane). After electrophoresis, proteins in the gel were transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). After blocking with 0.1% T-TBS containing 5% skim milk (Wako) at room temperature, the membrane was incubated with a mouse monoclonal anti-Aβ antibody (1:1000, clone 6E10, BioLegend, San Diego, CA, USA) or mouse monoclonal anti-GAPDH (1:10000, clone 5A12, Wako) in Can Get Signal solution 1 (Toyobo, Osaka, Japan) overnight at 4 °C. After washing with 0.1% T-TBS, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody against mouse IgG (1:2000; Cat. No. sc-2005, Santa Cruz) in Can Get Signal Solution 2 (Toyobo) for 2 h at room temperature. After washing, chemiluminescence on the membrane was detected by ECL Prime Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA) using an ImageQuant LAS 4000 system (GE Healthcare).
9
UV Cross-linking of Protein-RNA Complexes
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Proteins (200 nM) were incubated with 100 nM RNA (A20) labeled with internal 4-thiouridine (4SU) in the indicated position in a buffer containing 20 mM Tris, pH 7.0, 50 mM NaCl, 5 mM BME, 1 U/µL RNase inhibitor, human placenta (New England Biolabs), 1.5 mM MgCl2, and 1 mM AMPPNP (pH 7.0) at 22 °C for 30 min. Cross-linking was induced by exposure of the protein–RNA mixture to 365 nm UV light using a 4-W handheld UV lamp (UVP) for 20 min at 4 °C. Samples were quenched with NuPAGE lithium dodecyl sulfate sample buffer (Thermo Fisher Scientific) and subjected to SDS/PAGE analysis using NuPAGE 4 to 12% Bis-Tris protein gels (Thermo Fisher Scientific). Gels were scanned using a Typhoon FLA 9500 laser scanner. The 5′ fluorescein-labeled oligonucleotides used included the following 5′-to-3′ sequences: AAA AAA AAA AAA AAA AAA (4SU)A, AAA AAA AAA AAA AAA A(4SU)A AA, AAA AAA AAA AAA (4SU)AA AAA AA, AAA AAA AA(4SU) AAA AAA AAA AA, AAA A(4SU)A AAA AAA AAA AAA AA, and (4SU)AA AAA AAA AAA AAA AAA AA.
10
Quantification of GPR14 and GAPDH Proteins
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Protein levels of GPR14 and GAPDH were assessed in fresh frozen LV and rat isolated cardiomyocyte. Tissue and cells homogenized in lysis buffer (Cell Signaling Technology Inc., Danvers, MA) containing 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich Co., LLC., St. Louis, MO) was centrifuged and protein was quantified by the bicinchoninic acid assay (Thermo Fisher Scientific); NuPAGE lithium dodecyl sulfate sample buffer (Thermo Fisher Scientific) was added and lysates were electrophoresed on NuPAGE 4%–12% Bis-Tris polyacrylamide gels (Thermo Fisher Scientific). Proteins were transferred to polyvinylidene difluoride membranes and incubated with primary antibodies, anti-GPR14 (1:200; Santa Cruz Biotechnology, Inc., Dallas, TX), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:10,000; Cell Signaling Technology Inc.), followed by horseradish peroxidase-conjugated secondary antibodies (goat anti-mouse IgG1; Santa Cruz Biotechnology). Each sample was detected using ECL Advanced Western blotting detection kit (GE Healthcare UK Ltd., Little Chalfont, UK) and a chemiluminescence system. Band intensity was quantified using ImageJ software.
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