DNA of KIMAT1 full length or deletion constructs were amplified by PCR. T7 RNA polymerase promoter sequences were added to forward primers for subsequent in vitro transcription. PCR products were purified with Gel Extraction Kit (Qiagen) and transcribed in vitro using Biotin RNA labeling Mix Kit (Roche) and T7 RNA Transcription Polymerase (Roche) according to the manufacture’s instruction. 3 μg of Biotin-labeled lncRNA (full-length or fragments) were diluted into 40 μl RNA structure buffer (10 mM Tris-HCl pH7.0, 10 mM MgCl2, 0.1 M KCl), heated at 90 °C for 2 min and placed in ice for additional 2 min. Samples were kept at room temperature for 30 min to generate RNA secondary structures. Meanwhile, 80% confluent cells were washed with cold PBS and collected. After centrifugation, cells were re-suspended in 1 ml chilled RIP buffer (25 mM Tris-HCl pH7.4, 150 mM KCl, 5 mM EDTA, 0.5% NP40) and sheared using a Bioruptor device with 30 strokes of 30 s on and 45 s off. 2 mg of sheared lysates were added to the folded RNA in RIP buffer supplemented with a final concentration of 0.1 μg/μl tRNA at 4 °C for 2 h. Pre-washed streptavidin beads were mixed with RNA-cell lysate complex and further incubated for 1 h at 4 °C. Beads were washed six times and boiled in 1× Laemmli loading buffer (Bio-Rad). Retrieved protein samples were examined using immunoblotting.
Laemmli loading buffer
Manufactured by Bio-Rad
Sourced in United States
Laemmli loading buffer is a pre-formulated solution used to prepare protein samples for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It contains a mixture of chemicals that denature and solubilize proteins, allowing them to migrate through the gel based on their molecular weight.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (7 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Sds page gel, by Bio-Rad (5 mentions)
β mercaptoethanol, by Bio-Rad (5 mentions)
Ripa buffer, by Thermo Fisher Scientific (5 mentions)
β mercaptoethanol, by Merck Group (4 mentions)
Kpl detector block solution, by LGC (4 mentions)
Protease inhibitor, by Roche (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Gradient tris glycine gel, by Thermo Fisher Scientific (4 mentions)
Bca protein assay, by Thermo Fisher Scientific (4 mentions)
Tgx gel, by Bio-Rad (4 mentions)
Coomassie blue, by Thermo Fisher Scientific (3 mentions)
Mini protean tgx precast protein gel, by Bio-Rad (3 mentions)
Ap conjugate substrate kit, by Bio-Rad (3 mentions)
Protease inhibitor, by Merck Group (3 mentions)
Bca assay, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 680, by Thermo Fisher Scientific (3 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (3 mentions)
Clarity western ecl substrate, by Bio-Rad (3 mentions)
66 protocols using laemmli loading buffer
1
Biotin-labeled lncRNA-Protein Interactome
2
Protein Extraction and Western Blot Analysis
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Adherent cells were detached with trypsin (0.4% w/v solution, Gibco) and lysed on ice with RIPA buffer (150 mM NaCl, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0), 1× Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific). After incubating on ice for 40 min, the samples were centrifuged for 20 min at 15 000g, 4°C. Protein concentration of the supernatants was determined using the DC protein assay kit (Bio-Rad). Protein samples were prepared in 1× Laemmli loading buffer (Bio-Rad) with DTT and resolved on 7%, 8% or 12% Mini-PROTEAN TGX™ Precast Gels (Bio-Rad), using Tris/Glycine/SDS running buffer (Bio-Rad). After electrophoresis, proteins were transferred to low-fluorescence PVDF transfer membranes (Thermo Scientific) and blocked with 3% bovine serum albumin (BSA) in PBS, 0.1% Tween for 1 h at room temperature. Membranes were incubated overnight with primary antibodies at 4°C (Supplementary Table 2 ) in PBS with 3% BSA and 0.1% Tween, at 4°C and, after washing, incubated with the appropriate secondary antibody (Supplementary Table 2 ) for 1 h at room temperature. Proteins were detected using SuperSignalTM West Pico PLUS chemiluminiscent substrate (Thermo Scientific) and immunoblots were acquired via an iBright FL1500 Imaging System and quantified with iBright Analysis Software.
3
Functional Domains of SAA-RAGE Interaction
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Co-Immunoprecipitation (Co-IP) was used to investigate the functional domains of SAA responsible for receptor RAGE binding and subsequent endothelial dysfunction using the decoy target, esRAGE. Equal concentrations of SAA and esRAGE (1 µg/mL in complete media) were combined and incubated at 4 °C overnight with gentle rotary agitation. Immunolabelling was performed by adding 2 µg/mL polyclonal rabbit anti-human esRAGE antibody (final dilution 1:200 v/v) or IP Wash Buffer (containing 0.025 M Tris, 0.15 M NaCl, 0.1 mM EDTA, 1% (v/v) NP-40, 5% v/v glycerol, pH 7.4) alone (Control). The vials were then incubated for 2 h at 20 °C under rotary agitation. Protein G-Sepharose conjugated beads (GE™ Healthcare, Sydney, Australia, 100 µL) were added to each sample and mixed with rotary agitation for 1 h at 20 °C. Mixtures were then centrifuged at 2000× g for 1 min at 4 °C and the supernatant removed and discarded. The IP complexes were washed 3 times in IP Wash Buffer for 2 min by centrifugation and then resuspended in 2× Laemmli loading buffer (BioRad, Sydney, Australia). The complexes were boiled rapidly for 5 min at 95 °C to allow protein elution. Vials were centrifuged at 14,000× g for 1 min and the supernatant retained for immediate SDS-polyacrylamide gel electrophoresis.
4
Quantifying RhoA and Rac1 Activation
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Rhotekin-RBD (rho binding domain) beads (Cytoskeleton, Denver, CO, USA) were used to precipitate activated GTP-bound RhoA from retinal samples. To precipitate active GTP-bound RAC1, the p21 binding domain of Pak3 (PAK-GST-PBD)25 (link) expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins and immobilized by binding to glutathione-Sepharose 4B beads (Thermo Scientific) was used. Retinal samples were lysed as described above (in Western Blotting) and immediately added to 40 μg of Rhotekin-RBD beads or 60 μg of PAK-GST-PBD beads. The mixture was incubated for 1 hour at 4°C, then centrifuged for 2 minutes at 5000 rpm at 4°C, washed once with excess lysis buffer and twice with phosphate buffer saline (PBS), resuspended in 40 μL of 2× Laemmli loading buffer (Bio-Rad), boiled for 3 minutes, and stored at −20°C. RhoA and RAC1 activation assay samples and total retinal protein were analyzed by standard Western blot techniques.
5
Assessing Recombinant Protein Integrity
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To determine protein integrity, 1.5 ug of the respective recombinant N1 mutant protein was mixed 1:1 with 2× Laemmli loading buffer (Bio-Rad). For reducing conditions, loading buffer was supplemented with 5% beta-mercaptoethanol. To assess the extent of protein multimerization, samples were treated with the crosslinker bis-sulfosuccinimidyl suberate (BS3, ThermoFisher, Waltham, MA, USA) according to the manufacturer’s instructions. Bovine serum albumin (BSA) was used as a monomeric control and recombinant A/Michigan/45/2015 N1 protein containing only the globular head domain and the VASP tetramerization domain was used as control for tetramerization. Samples were heated at 95°C for 15 min prior to loading them on a sodium dodecyl sulphate polyacrylamide gel (4–20% Mini-PROTEAN® TGX™ Precast Protein Gels, BioRad). Gels were stained with Coomassie blue (ThermoFisher) for 1 h at RT and de-stained with distilled water to visualize the proteins.
6
Streptavidin Bead Elution and In-Gel Tryptic Digestion
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For the APEX2 experiments, streptavidin beads were eluted by boiling with 2× Laemmli loading buffer (Biorad, 161 to 0737) containing 20 mM DTT and 2 mM biotin. The eluate was then run on a 4% to 15% Tris-glycine gel (Biorad, #4561084) for 30 min at 50 V. Gel slices were then fixed according to Mackinder et al. (2017) (link). In-gel tryptic digestion was performed after reduction with 10 mM dithioerythritol and 50 mM S-carbamidomethylation with iodoacetamide. Gel pieces were washed 2 times with aqueous 50% (v/v) acetonitrile containing 25 mM ammonium bicarbonate and then once with acetonitrile and dried in a vacuum concentrator for 20 min. A 500-ng aliquot of sequencing-grade trypsin (Promega) was added prior to incubation at 37 °C for 16 h.
7
Copper-Catalyzed Protein Labeling Assay
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Confluent
THP-1 cells were washed twice with 1× PBS (pH 7.4)
and then lysed in 1× PBS (pH 7.4) with 0.1% NP-40 and mammalian
protease inhibitors (MilliporeSigma, St. Louis, MO), using a sonicator
set to 30% amplitude with six 10 s pulses. After sonication, the lysates
were clarified by centrifugation at 10 000g for 15 min. Protein concentration was determined using the Bio-Rad
Protein Assay. The cell lysates were diluted to 2 mg/mL, and1 , 2 , or DMSO was added to a concentration of
100 μM or 0.1% (DMSO). After incubation for 1 h, the reagents
for CuAAC were added to final concentrations of 10 mM CuSO4, 1 mM TCEP, and 1 mM TBTA. The reaction was incubated at 4 °C
for at least 1 h. Following incubation, the proteins were precipitated
with 4 volumes of methanol and 1 volume of chloroform. This precipitate
was pelleted by centrifugation at 4000g for 15 min.
The pellet was resuspended in 2× Laemmli loading buffer (Bio-Rad)
and loaded directly into a Tris–glycine 10% polyacylamide SDS-PAGE
gel without heating or reducing agent. Immunoblotting proceeded as
described above.
THP-1 cells were washed twice with 1× PBS (pH 7.4)
and then lysed in 1× PBS (pH 7.4) with 0.1% NP-40 and mammalian
protease inhibitors (MilliporeSigma, St. Louis, MO), using a sonicator
set to 30% amplitude with six 10 s pulses. After sonication, the lysates
were clarified by centrifugation at 10 000g for 15 min. Protein concentration was determined using the Bio-Rad
Protein Assay. The cell lysates were diluted to 2 mg/mL, and
100 μM or 0.1% (DMSO). After incubation for 1 h, the reagents
for CuAAC were added to final concentrations of 10 mM CuSO4, 1 mM TCEP, and 1 mM TBTA. The reaction was incubated at 4 °C
for at least 1 h. Following incubation, the proteins were precipitated
with 4 volumes of methanol and 1 volume of chloroform. This precipitate
was pelleted by centrifugation at 4000g for 15 min.
The pellet was resuspended in 2× Laemmli loading buffer (Bio-Rad)
and loaded directly into a Tris–glycine 10% polyacylamide SDS-PAGE
gel without heating or reducing agent. Immunoblotting proceeded as
described above.
8
Immunoprecipitation of OPRS1 and IP3R1 Proteins
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The appropriate monoclonal (3 µg) or polyclonal antibody (6 µg) was incubated with 60 µl of washed magnetic beads (Dynabeads M-280 coated with sheep anti-mouse IgG or M-280 coated with sheep anti-rabbit IgG (Life Technologies, Dynal AS, Norway)) overnight at 4 °C on a rotator (VWR International, LLC, PA, USA). The beads with attached antibodies were washed twice (200 µl) with phosphate-buffered saline (PBS supplemented with 1% bovine serum albumin). Proteins were immunoprecipitated from 1 mg of detergent-extracted total protein via their incubation with antibody-bound beads for 4 h at 4 °C. Bead complexes were washed with PTA (4× with 200 µl; 145 mmol/L NaCl, 10 mmol/L NaH2PO4, 10 mmol/L sodium azide, and 0.5% Tween 20; pH 7.0). Immunoprecipitated proteins were then extracted with 60 µl of 2× Laemmli loading buffer according to the manufacturer’s instructions (Bio-Rad) and boiled for 5 min. The following antibodies were used for immunoprecipitation: rabbit polyclonal antibody to OPRS1 (σ1R; AB_881796, Abcam, UK) and mouse monoclonal antibody to IP3R1 (AB_212025, Calbiochem, Merck Biosciences, Germany).
9
Western Blot Analysis of ALV-J SU Protein
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Transfected DF-1 cells were washed with phosphate-buffered saline and then lysed in 2× Laemmli loading buffer (Bio-RAD). Proteins were boiled for 5 min, and then were separated by SDS-polyacrylamide gel electrophoresis (PAGE). After SDS-PAGE, the proteins were transferred onto nitrocellulose membrane. The membrane was stained with 2.5% fast green in 10% acetic acid for 2 min to visualize blotted proteins. Destaining was performed with 10% acetic acid for 10 min. The membrane was blocked with 10% skim milk. The ALV-J SU protein was detected using the 1D4 monoclonal antibody against ALV-J (dilution, 1:1000). For detection of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a monoclonal antibody (Ambion) was used at a dilution of 1:4,000. Peroxidase-coupled secondary antibodies directed against mouse immunoglobulin (Pierce) were diluted 1:10,000.
10
Western Blot Analysis of ER Stress Markers
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Cell lysates were collected using RIPA buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After heated at 75°C for 20 minutes, the samples were stored at −20°C. Before running, samples were sonicated and mixed with 2× Laemmli loading buffer (BIO-RAD, Hercules, CA, USA) and then heated at 95°C for 5 minutes. Each sample (30 μL) was loaded to 8% to 16% Tris-glycine gel (Invitrogen, Carlsbad, CA, USA), and electrophoresis was performed for 1 to 2 hours at 120 V. Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and incubated with Odyssey blocking buffer (LI-COR Biotechnology, Lincoln, NE, USA) for 1 hour at room temperature. The membrane was incubated with primary antibodies diluted in blocking buffer with 0.01% Tween-20 at 4°C overnight. Antibodies for GRP78 (BIP; 1:1000) and PDI (1:500) were purchased from Cell Signaling Technology. β-Actin antibody (1:500) was purchased from BioLegend, Inc. (San Diego, CA, USA). Then, membranes were incubated with goat anti-mouse and goat anti-rabbit secondary antibodies (IRDye 680LT, IRDye 800CW; LI-COR Biosciences). The fluorescent signals were captured on an infrared imager (Odyssey Infrared Imager; LI-COR Biosciences).
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