HEK293T cells growing in 15-cm culture dishes and transiently expressing 1D4-tagged hSERT mutants were suspended in PBS 40–48 h post-transfection, pelleted by centrifugation (700g, 5 min) and solubilized (2 h, 4 °C) in solubilization buffer (50 mM Tris-HCl, pH 7.5, 145 mM NaCl, 5 mM EDTA and 1% Triton X-100 or IGEPAL CA-630, supplemented with protease inhibitors (cOmplete, Roche)). After centrifugation (18,000g, 30 min), the supernatant fraction was incubated overnight at 4 °C with Dynabeads Protein G (Life Technologies) bound to Rho1D4 mAb or with sepharose 2B beads conjugated to Rho1D4 mAb. Beads were washed five times with solubilization buffer and Dynabeads Protein G bound samples were eluted for 10 min at 70 °C with LDS loading buffer (Life Technologies), while sepharose bead samples were eluted twice for 20 min at 60 °C with LDS loading buffer.
Lds loading buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
LDS loading buffer is a buffer solution used in electrophoresis techniques to prepare samples for analysis. It helps maintain the structure of proteins and facilitates their separation based on their molecular weight.
Automatically generated - may contain errors
Lab products found in correlation
Bis tris polyacrylamide gel, by Thermo Fisher Scientific (8 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Luminata forte, by Merck Group (4 mentions)
Anti ha agarose antibody beads, by Merck Group (4 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (3 mentions)
Supersignal west femto, by Thermo Fisher Scientific (3 mentions)
Nupage transfer buffer, by Thermo Fisher Scientific (3 mentions)
Supersignal west femto chemiluminescence kit, by Thermo Fisher Scientific (3 mentions)
Nitrocellulose membrane, by GE Healthcare (3 mentions)
Nupage gel, by Thermo Fisher Scientific (3 mentions)
Trichloroacetic acid, by Merck Group (3 mentions)
Mouse anti gapdh, by Novus Biologicals (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Ecl western blotting substrate, by Thermo Fisher Scientific (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Bis trispolyacrylamide gels, by Bio-Rad (2 mentions)
Sypro ruby protein gel stain, by Bio-Rad (2 mentions)
Image lab 6, by Bio-Rad (2 mentions)
Heat inactivated bovine serum, by Thermo Fisher Scientific (2 mentions)
95 protocols using lds loading buffer
1
Immunopurification of Recombinant SERT Mutants
2
In vitro UBE2Q1 autoubiquitylation assay
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In vitro UBE2Q1 autoubiquitylation assay was performed as previously described (32 (link)). Briefly, the reaction was started by combining an equal volume of ubiquitin solution (40 μM) with an enzymatic mixture consisting of UBE1 (1.0 μM), ATP (5 mM), MgCl2 (20 mM), UBE2Q1 full-length or minimal catalytic domain (5.0 μM) in 1× PBS buffer (pH 7.5), and 0.5 mM TCEP. The reaction was incubated at 30°C for 45 min.
Compound 1 E1 inhibitor (MLN7243) was added at a final concentration of 10 μM, and the reaction was incubated for a further 15 min at room temperature. The reaction was then subaliquoted and treated with either JOSD1 (3.0 μM), USP2 (3.0 μM), or 1× PBS buffer for 30 min at 30°C. Reactions were stopped by adding 1× final lithium dodecyl sulfate–loading buffer (4× LDS loading buffer, Thermo Fisher Scientific) with or without β-mercaptoethanol and supplemented with either 1× PBS or hydroxylamine (0.5 M) or NaOH (0.5 M).
The samples were incubated for 30 min at 37°C, and the reaction mixtures were visualized using the 4 to 12% gradient SDS–polyacrylamide gel electrophoresis (SDS-PAGE) gels. The images were captured using the ChemiDoc Imaging System (Bio-Rad).
Compound 1 E1 inhibitor (MLN7243) was added at a final concentration of 10 μM, and the reaction was incubated for a further 15 min at room temperature. The reaction was then subaliquoted and treated with either JOSD1 (3.0 μM), USP2 (3.0 μM), or 1× PBS buffer for 30 min at 30°C. Reactions were stopped by adding 1× final lithium dodecyl sulfate–loading buffer (4× LDS loading buffer, Thermo Fisher Scientific) with or without β-mercaptoethanol and supplemented with either 1× PBS or hydroxylamine (0.5 M) or NaOH (0.5 M).
The samples were incubated for 30 min at 37°C, and the reaction mixtures were visualized using the 4 to 12% gradient SDS–polyacrylamide gel electrophoresis (SDS-PAGE) gels. The images were captured using the ChemiDoc Imaging System (Bio-Rad).
3
Quantitative Western Blot Analysis
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Synchronised populations were grown on PCK-NGM plates until the young adult stage and washed off plates using M9 buffer supplemented with 0.001% Triton-X100. Worms were settled, supernatant was removed, and worms were resuspended in undiluted 4× LDS loading buffer (Thermo Fisher Scientific) supplemented with NuPAGE Sample Reducing Agent (Thermo Fisher Scientific). Lysis was performed by a freeze/thaw cycle followed by 10 min incubation at 95°C.
Samples were run on precast Bolt 4–12% gels (Thermo Fisher Scientific) for 19 min at 200 V. Proteins were transferred from the gel onto a nitrocellulose membrane using an iBlot2 device (Thermo Fisher Scientific).
After transfer, the membrane was blocked using 5% milk powder in PBST (PBS + 0.1% Tween-20) for 1 hr at room temperature. Incubation with primary antibodies was carried out in PBST + 5% milk powder at 4°C overnight. Blots were washed 6 × 5 min with PBST + 5% milk powder before incubating with secondary antibody for 1 hr at room temperature.
Antibodies used are listed in the key resources table. Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) or SuperSignal West Femto chemiluminescent Substrate (Thermo Fisher Scientific) were used as detection agent. For quantitative blots, a C-DiGit Blot Scanner (LI-COR) was used, and intensities were analysed using ImageStudio software.
Samples were run on precast Bolt 4–12% gels (Thermo Fisher Scientific) for 19 min at 200 V. Proteins were transferred from the gel onto a nitrocellulose membrane using an iBlot2 device (Thermo Fisher Scientific).
After transfer, the membrane was blocked using 5% milk powder in PBST (PBS + 0.1% Tween-20) for 1 hr at room temperature. Incubation with primary antibodies was carried out in PBST + 5% milk powder at 4°C overnight. Blots were washed 6 × 5 min with PBST + 5% milk powder before incubating with secondary antibody for 1 hr at room temperature.
Antibodies used are listed in the key resources table. Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) or SuperSignal West Femto chemiluminescent Substrate (Thermo Fisher Scientific) were used as detection agent. For quantitative blots, a C-DiGit Blot Scanner (LI-COR) was used, and intensities were analysed using ImageStudio software.
4
Measuring Nascent Protein Synthesis in Flies
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To measure nascent protein synthesis, 10 25-day heat-treated male flies were homogenized in 80 μL of cold solubilization buffer (15 mM Tris-HCl, 300 mM NaCl, 15 mM MgCl2, 2 mM DTT, 1% Triton X-100, and 12.5 μL/mL RNase In; pH 7.5) supplemented with 100 μM puromycin. Lysates were incubated at 4 °C for 5 min and mixed with 40 μL of 4× LDS loading buffer (Thermo Fisher Scientific, Waltham, MA, USA). Homogenized samples were then boiled for 8 min and centrifuged at 20,000× g for 30 min. To separate the supernatant, 4–12% gradient SDS-PAGE (Invitrogen, Carlsbad, CA, USA) was used, which was transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA). The membrane was incubated with mouse anti-puromycin antibody (1:10000; Millipore, Burlington, MA, USA, MABE343) overnight and was detected with HRP-conjugated mouse secondary antibodies (1:10000; Millipore, Burlington, MA, USA, AP308P). Detection was carried out using an ECL-Plus kit (Thermo Fisher Scientific, Waltham, MA, USA).
5
E2 Ubiquitin Charging Assay
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WT or mutant E2s (10 μM) were charged with labeled Ub (Cy3/Cy3b/Cy5) (12.5 μM) as above. An equal volume of a sample containing 20 mM Na2PO4pH ∼7.5, 20 mM l -lysine, or buffer alone for control reactions, was added and a time point was taken at t = 0. Further samples were taken at the indicated time points and quenched with 4× LDS loading buffer (ThermoFisher).
6
PNA Inhibition of RickA Protein Expression
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The region starting 100 base pairs upstream of the start codon through 600 base pairs downstream of the rickA gene was amplifed using rickA forward primer 5’- ATTCGTTCATCTATCTTTTTTTTATTTATC-3’ and reverse primer 5’-TTTTTGTATTTCTTTAAGTTCTTTGACATTAG-3’ and cloned into pEXP5-CT (Life Technologies, Grand Island, NY). Translation was performed using the Expressway Cell-Free E. coli Expression System (Life Technologies). PNA targeted to rickA was added to the reaction to demonstrate PNA specificity for its target sequence as measured by impaired protein production. The CALML3 control vector was included in each reaction to serve as a loading control and to demonstrate PNA specificity. The total volume of each sample was resuspended in LDS loading buffer (Life Technologies) with reducing agent, heated for 10 minutes at 70°C and run on NuPAGE 4–12% Bis-Tris gels. Gels were transferred to PVDF membranes using an iBlot gel transfer device (Life Technologies). Membranes were probed using an anti-His antibody (1:3000) (Thermo Fisher Scientific, Waltham, MA) and developed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Inc.).
7
Comparative Proteomic Analysis of Stem/Progenitor Cells
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Equal numbers of cells from each stem or progenitor population were sorted into HBSS supplemented with 2% (v/v) heat-inactivated bovine serum (GIBCO). Samples were washed once with PBS and centrifuged at 2500 g at 4°C for 5 min. Supernatant was discarded and protein pellets were directly lysed and solubilized in 9M urea, 2% SDS, 50 mM DTT, and 50 mM Tris pH 7.4. After incubating the samples for 10–15 min at room temperature, LDS loading buffer (Life Technologies) was added and the sample was heated at 70°C for 10 minutes. Samples were separated on Bis-Trispolyacrylamide gels (Life Technologies). The Bis-Trispolyacrylamide gels were then stained with SYPRO Ruby Protein Gel Stain (Bio-Rad) for 3 h and washed in a solution containing 7% acetic acid and 10% methanol for 1 h. Gels were imaged using Gel Doc XR (Bio-Rad) and analyzed with Image Lab 6.0.1 software (Bio-Rad).
For bone marrow cells, incubated at 37°C or 42°C, 105 cells were sorted into TCA. The final concentration was adjusted to 10% TCA. Extracts were incubated on ice for at least 15 min and centrifuged at 15,000 g at 4°C for 15 min. Precipitates were washed in acetone twice and dried. The pellets were solubilized in 6 M urea, 2% (v/v) Triton X-100, and 1% (v/v) 2-mercaptoethanol. The protein content in the supernatant was assessed with the microBCA assay (Pierce).
For bone marrow cells, incubated at 37°C or 42°C, 105 cells were sorted into TCA. The final concentration was adjusted to 10% TCA. Extracts were incubated on ice for at least 15 min and centrifuged at 15,000 g at 4°C for 15 min. Precipitates were washed in acetone twice and dried. The pellets were solubilized in 6 M urea, 2% (v/v) Triton X-100, and 1% (v/v) 2-mercaptoethanol. The protein content in the supernatant was assessed with the microBCA assay (Pierce).
8
Proteome-wide Detection of Posttranslational Modifications
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Equal numbers of cells from each stem or progenitor population were sorted into trichloracetic acid (TCA, Sigma). The final concentration was adjusted to 10% (v/v) TCA. Extracts were incubated on ice for at least 15 min and centrifuged at 15,000 g at 4°C for 15 min. Precipitates were washed in acetone twice and dried. The pellets were solubilized in 9 M urea, 2% (v/v) Triton X-100, and 1% DTT. LDS loading buffer (Life Technologies) was added and the pellet was heated at 70°C for 10 min. Samples were separated on Bis-Trispolyacrylamide gels (Life Technologies) and transferred to PVDF membranes (Bio-Rad). Western blot analyses were performed according to the protocol from Cell Signaling Technologies. Blots were stripped with 1% SDS, 25 mM glycine (pH 2) before re-probing. The following primary and secondary antibodies were used for western blot analyses: Ubiquitin (P4D1; Cell Signaling), K48-Ubiquitin (polyclonal; Cell Signaling 4289), c-Myc (polyclonal; Cell Signaling 9402S), phos-Eif2α (S51; Cell Signaling) β-Actin (AC74; Sigma), Gapdh (14C10; Cell Signaling), HRP-linked anti-rabbit IgG (Cell Signaling) and HRP-linked anti-mouse IgG (Cell Signaling). Blots were developed with the SuperSignal West Femto or Pico PLUS chemiluminescence kit (Thermo Scientific).
9
Protein-Protein Interaction Assay in HEK293 Cells
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HEK293 cells stably expressing hemagglutinin (HA)-tagged PTHR and cultured on a 10-cm dish were stimulated with PTH(1–34)-NH2 or analogues at 100 nM for 5 min. Cells were then washed with ice-cold PBS prior to crosslinking for 2 h with dithiobis (succinimidyl propionate) DSP (Covachem, #13301) in PBS at 4 °C. The reaction was stopped by addition of 10 mM Tris–HCl for 10 min and cell lysates were prepared using lysis buffer (1% Triton X-100, 50 mM Tris–HCl pH 7.4, 140 mM NaCl, 0.5 mM EDTA) containing protease and phosphatase inhibitors (Roche, #11873580001). Protein concentration was determined using BCA protein assay kit (ThermoFisher, #23225), and lysates were incubated with anti-HA agarose antibody beads (Sigma-Aldrich; #A2095 clone HA-7) overnight at 4 °C. Elution was done using LDS loading buffer (Life Technologies) and samples were loaded on 10% SDS-PAGE and transferred to nitrocellulose membrane. We used primary antibodies against HA (Covance, clone 16B12, Mouse IgG1) and β-arrestin1/2 (Cell Signaling; #4674, clone D24H9, Rabbit IgG); anti-Mouse HRP and anti-Rabbit HRP (Dako, Goat polyclonal) secondary antibodies were then used. Immunoreactive bands were visualized with Luminata Forte (EMD Millipore) and autoradiography film.
10
Purification and Characterization of Membrane Proteins
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Isopropyl-β-d -thiogalactopyranoside (IPTG), Tris(2-carboxyethyl)phosphine (TCEP), n-dodecyl-β-d -maltopyranoside (DDM) and precast SDS–PAGE gels were from Generon. All aromatic acids, l -arabinose, cholesteryl hemisuccinate Tris salt (CHS), 0.4–0.6 mm acid-washed glass beads, valinomycin and Proteinase K-agarose were from Sigma–Aldrich. HisTrap columns, PD-10 and PD SpinTrap G-25 gel filtration columns, Superdex 200 10/300 GL column, size exclusion column standards, and nitrocellulose membrane were from GE Healthcare. Chemiluminescence reagents (LumiGLO) were from Cell Signaling Technologies. E. coli strain BL21-AI, LDS loading buffer, V5-HRP, pyranine and gels and reagents for blue native PAGE were from Life Technologies. Centrifugal concentrators were from Millipore. The detergent compatible Lowry assay was purchased in kit format from ThermoFisher. E. coli polar lipid extract and egg phosphatidylcholine were from Avanti Polar Lipids.
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