Protein loading buffer

Manufactured by Bio-Rad

Sourced in United States

Protein loading buffer is a solution used to prepare protein samples for electrophoresis. It helps maintain the structure and charge of proteins, ensuring they can be effectively separated and analyzed.

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Lab products found in correlation

Bca protein assay kit, by Thermo Fisher Scientific (4 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions) Insulin, by Merck Group (2 mentions) Dexamethasone (dex), by Merck Group (2 mentions) Clarity western ecl substrate, by Bio-Rad (2 mentions) Pvdf membrane, by Merck Group (1 mentions) Chemidoc mp system, by Bio-Rad (1 mentions) Western lightning plus ecl, by PerkinElmer (1 mentions) Cocktail of protease inhibitor, by Merck Group (1 mentions) Protein a g beads, by Merck Group (1 mentions) Bicinchoninic acid method, by Bio-Rad (1 mentions) Enhanced chemiluminescence system, by Bio-Rad (1 mentions) Polyvinylidene difluoride membrane, by Merck Group (1 mentions) Horseradish peroxidase conjugated secondary antibody, by Merck Group (1 mentions) Pierce radioimmunoprecipitation assay buffer, by Thermo Fisher Scientific (1 mentions) Coomassie blue r 250, by Bio-Rad (1 mentions) Perilipin, by Cell Signaling Technology (1 mentions) Phospho pka substrate, by Cell Signaling Technology (1 mentions) Antibody against β actin, by Abcam (1 mentions) Rosiglitazone rosi, by Merck Group (1 mentions)

12 protocols using protein loading buffer

Western Blot Protein Analysis Protocol

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Proteins from total cell lysate or protein samples from in vitro pull-down or co-IP denatured in protein loading buffer (Bio-Rad) were resolved on 10% SDS-PAGE gel and then transferred to PVDF membrane (Millipore). The membrane was blocked by 5% non-fat milk in TBS-tween 20 (TBST) buffer for 1 h at room temperature. The membrane was then incubated with primary antibodies at 4 °C overnight with mild shaking. After washing three times with TBST buffer, the membrane was incubated with HRP-conjugated goat anti-mouse or anti-rabbit secondary antibody (GE Healthcare) for 1 h at room temperature. The protein bands were then detected by Western Lightning Plus-ECL (Perkin Elmer) after washing with TBST three times. The membrane was imaged on the ChemiDoc MP system (Bio-Rad).

Du Y., Yan Y., Xie S., Huang H., Wang X., Ng R.K., Zhou M.M, & Qian C. (2021). Structural mechanism of bivalent histone H3K4me3K9me3 recognition by the Spindlin1/C11orf84 complex in rRNA transcription activation. Nature Communications, 12, 949.

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Protein Interaction Immunoprecipitation

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Cells were collected after transfected with indicated plasmids. After washing with 1 × PBS, cells were lysed in IP buffer (0.5% NP-40, 20 mM Tris-HCl, pH = 8.0, 10 mM NaCl, 1 mM EDTA) supplied with a cocktail of protease inhibitors (Sigma-Aldrich). Cell lysate was immunoprecipitated with indicated antibodies against target proteins in the presence of protein A/G beads (Millipore) overnight. Beads were washed with IP buffer for five times and boiled in protein loading buffer (Bio-Rad) for further analysis by Western blot.

Ding D., Zheng R., Tian Y., Jimenez R., Hou X., Weroha S.J., Wang L., Shi L, & Huang H. (2022). Retinoblastoma protein as an intrinsic BRD4 inhibitor modulates small molecule BET inhibitor sensitivity in cancer. Nature Communications, 13, 6311.

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Western Blot Protein Analysis Protocol

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Protein lysates were extracted from cells using the Pierce radio immunoprecipitation assay buffer (Thermo Fisher) with 1% protease inhibitor. Protein lysates were quantified using the bicinchoninic acid method (Biorad). Quantified protein lysates were diluted in protein loading buffer (Biorad) and denatured at 95°C for 10 min. The protein samples were then resolved on sodium dodecyl sulfate–
polyacrylamide gel electrophoresis, transferred onto a polyvinylidene difluoride membrane (Millipore), and then blocked with 5% BSA in Tris‐buffered saline‐Tween 20 (TBS‐T; Sigma) for 1 h at room temperature. The blocked membrane was incubated with primary antibodies in 5% BSA in TBS‐T at 4°C overnight. All antibodies used for western blotting are listed in Table S1. After washing with TBS‐T, the membrane was incubated for 1 h with horseradish peroxidase‐conjugated secondary antibody (1:3000; Sigma). A complex of primary and secondary antibody‐labeled proteins was detected by an enhanced chemiluminescence system (Biorad) followed by manual exposure using the Hypercassette Autoradiography Cassette (Cytiva Amersham).

Gong L., Zhang Y., Yang Y., Yan Q., Ren J., Luo J., Tiu Y.C., Fang X., Liu B., Lam R.H., Lam K., Lee A.W, & Guan X. (2022). Inhibition of lysyl oxidase‐like 2 overcomes adhesion‐dependent drug resistance in the collagen‐enriched liver cancer microenvironment. Hepatology Communications, 6(11), 3194-3211.

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Affinity Purification of miRNA Complexes

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To further confirm the direct interaction, DIG-conjugated miR-200c or let-7a (Takara Biomed) was incubated with whole cell extracts of 4T1 lung metastasis, then the miRNA complex was pulled down using anti-Dig Alpha Donor beads (PerkinElmer) in binding buffer (0.1% NP-40, 5 mM EDTA, 0.5 M NaCl, 0.01% SDS). After washing with PBS/0.01%SDS, the miR-200c or let-7a complex was eluted with the protein loading buffer (BioRad), and then analyzed by electrophoresis with 10% SDS-PAGE following an staining with Bio-Rad Coomassie Blue R-250 and standard Western blotting or MS analysis.

Liu Y., Tang J., Qiu X., Teng L.A., Sriwastva M.K., Han X., Li Z., Liu M., Liu S., Da D., Li Z., Zhen L, & Ren Y. (2023). Rab1A-Mediated Exosomal Sorting of miR-200c Enhances Breast Cancer Lung Metastasis. Breast Cancer : Targets and Therapy, 15, 403-419.

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Medicarpin Regulates Adipocyte Metabolism

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Medicarpin (purity 98%, HPLC) was purchased from ChemFaces Biochemical Co., Ltd. (Wuhan, Hubei, China). Dexamethasone, Insulin, isobutyl-1-metylxanthine (IBMX), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Isoproterenol (Iso) and rosiglitazone (Rosi) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) and High-glucose Dulbecco’s modified Eagle medium (DMEM) were obtained from Atlas Biologicals. Penicillin-streptomycin solution was obtained from Hyclone Laboratories, Inc. (South Logan, NY, USA). Antibodies against HSL, Phospho-HSL (Serine 563 and 660), ATGL, Phospho-PKA substrate and Perilipin were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibody against β-actin was purchased from Abcam (Cambridge, MA, USA). BCA protein assay kit was purchased from Thermo Scientific (Rockford, IL, USA) and protein loading buffer was obtained from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Bodipy 493/503 was obtained from Life Technologies Corporation (Carlsbad, CA, USA).

Imran K.M., Yoon D., Lee T.J, & Kim Y.S. (2018). Medicarpin induces lipolysis via activation of Protein Kinase A in brown adipocytes. BMB Reports, 51(5), 249-254.

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Insulin Signaling Pathway Modulation

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Ursolic acid, cytochalasin B, insulin, 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, wortmannin, SB203580, compound C, protease inhibitor and bovine serum albumin (BSA) were purchased from Sigma (St. Louis, MO, USA). High glucose Dulbecco’s modified Eagle’s medium (DMEM) was from Mediatech, Inc. (Cellgro Mediatech, Inc. Manassas, VA). Fetal bovine serum (FBS) was bought from PAA Laboratories (Etobicoke, ON, Canada). Bovine calf serum (BCS) was purchased from Cayman Chemical Company (Ann Arbor, Michigan, USA). The BCA protein assay kit was obtained from Thermo Scientific (San Jose, CA, USA). RIPA lysis buffer was from Millpore (MA, USA). Protein loading buffer was from Bio-Rad (Montreal, QC, Canada). Antibodies against phospho-phosphoinositide-dependent kinase (pPDK), phosphoinositide-dependent kinase (PDK), phospho-protein kinase C (PKC), protein kinase C (PKC), phospho-AS160 (pAS160), AS160, GLUT4, glucose transporter 1 (GLUT1), phospho-phosphoinositide-dependent serine/threonine kinase (pAKT), phosphoinositide-dependent serine/threonine kinase (AKT) and clathrin were from Cell Signaling Technology, Inc. (Beverly, Massachusetts, USA). 2-NBD-glucose was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). BM chemiluminescence blotting substrate kit was from Roche Diagnosis (Laval, QC, Canada).

He Y., Li W., Li Y., Zhang S., Wang Y, & Sun C. (2014). Ursolic Acid Increases Glucose Uptake through the PI3K Signaling Pathway in Adipocytes. PLoS ONE, 9(10), e110711.

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Western Blot Quantification of PD-L1

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The relative PD-L1
concentration in cell lysates was estimated using the Pierce BCA Protein
Assay Kit (Thermo Scientific). The samples (roughly 10 μg total
protein per well) were mixed with protein-loading buffer (Bio-Rad)
containing 2-mercaptoethanol. After denaturation by 5 min boiling,
the samples were loaded on 4–15% Mini-PROTEAN TGX precast gels
sodium dodecyl sulfate polyacrylamide gel (Bio-Rad). The gels were
blotted onto poly(vinylidene difluoride) (PVDF) membranes for 90 min
@ 80 V; proteins were transferred onto PVDF membranes and blocked
for nonspecific binding with Tris-buffered saline with Tween-20 (TBST)
[0.05% (v/v) Tween-20 in TBS pH 7.4] plus 5% bovine serum albumin
for 1 h at room temperature. PD-L1 RabMab antibodies (ab205921) (Abcam,
USA; at dilution of 1:800–1000) were applied overnight at 4
°C. The membrane was washed with TBST and then incubated with
horseradish peroxidase (HRP)-conjugated secondary antibodies (goat
antirabbit IgG H&L (HRP), ab97051) (Abcam, USA; at dilution 1:5000)
for 2 h. After washing, protein bands were visualized using Clarity
western ECL substrate (Bio-Rad) and analyzed using ImageJ v1.40 software.

Wu Y., Gu W., Chen C., Do S.T, & Xu Z.P. (2018). Optimization of Formulations Consisting of Layered Double Hydroxide Nanoparticles and Small Interfering RNA for Efficient Knockdown of the Target Gene. ACS Omega, 3(5), 4871-4877.

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Quantifying PD-1 Protein Levels in Cell Lysates

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The relative PD-1 concentration in cell lysates was estimated using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). The samples (roughly 10 µg total protein per well) were mixed with protein-loading buffer (Bio-Rad, Hercules, CA, USA) containing 2-mercaptoethanol. After denaturation by 5-min boiling, the samples were loaded on 4–15% Mini-PROTEAN^® TGX™ Precast Gels SDS polyacrylamide gel (Bio-Rad). Gels were blotted onto polyvinylidene difluoride (PVDF) membranes for 90 min at 80 V, the proteins were transferred onto PVDF membranes and then blocked for non-specific binding with TBST (0.05% (v/v) Tween-20 in TBS pH 7.4) plus 5% BSA for 1 h at RT. PD-1 RabMab antibodies (ab205921) (Abcam, Cambridge, UK; at dilution of 1:800–1000) was applied overnight at 4 °C. The membrane was washed with TBST and then incubated with HRP-conjugated secondary antibodies (Goat anti-rabbit IgG H&L (HRP), ab97051) (Abcam, Cambridge, UK; at dilution 1:5000) for 2 h. After washing, the protein bands were visualized using Clarity™ Western ECL Substrate (Bio-Rad) and then analyzed using ImageJ v1.40 software (Hercules, CA, USA).

Wu Y., Gu W., Li L., Chen C, & Xu Z.P. (2019). Enhancing PD-1 Gene Silence in T Lymphocytes by Comparing the Delivery Performance of Two Inorganic Nanoparticle Platforms. Nanomaterials, 9(2), 159.

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Cerebellar Protein Enrichment and Proteomic Analysis

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Streptavidin magnetic beads (Pierce) were used to enrich
biotinylated proteins from cerebellar lysates: 150 μL was used with
six 300 μm cerebellar slices for biochemistry experiments, and a
total of 400 μL was used for each proteomic sample. Calculation of
the estimated biotinylated/enriched protein amount based on bead usage and
bead binding capacity from the manufacturer (each 100 μL of bead
captures an estimated ~55 μg biotinylated rabbit IgG) suggests
that we captured ~220 μg proteins per proteomic sample in the
labeled/experimental groups. Beads were washed twice with normal RIPA buffer
and then incubated with the post-ultracentrifugation lysates on a 4°C
rotator overnight (14 hours). Beads were then sequentially washed twice with
1 mL normal RIPA buffer, once with 1 mL 1 M KCl, once with 1 mL 0.1 M
Na₂CO₃, once with 1 mL 2 M urea in 10 mM Tris-HCl
[pH 8.0], and twice with 1 mL normal RIPA buffer. For silver stain or
western blot, biotinylated proteins were eluted by heating the beads at
95°C for 10 minutes in 20 μL of 3x protein loading buffer
(Bio-Rad) supplemented with 20 mM dithiothreitol (DTT) and 2 mM biotin. For
proteomic samples, on-bead trypsin digestion was performed after enrichment
(see below for details). All chemicals were purchased from
Sigma-Aldrich.

Shuster S.A., Li J., Chon U., Sinantha-Hu M.C., Luginbuhl D.J., Udeshi N.D., Carey D.K., Takeo Y.H., Xie Q., Xu C., Mani D.R., Han S., Ting A.Y., Carr S.A, & Luo L. (2022). In situ cell-type-specific cell-surface proteomic profiling in mice. Neuron, 110(23), 3882-3896.e9.

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Magnetic Bead-Based Protein Enrichment and Preparation

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Streptavidin magnetic beads (Pierce) were used to enrich biotinylated proteins from brain lysates. For silver stain or Western blot, 20 μL was used for each sample from 50 dissected brains. For proteomic samples, 400 μL was used for each experimental group of 1000 dissected brains. Beads were first washed twice with normal RIPA buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.2% SDS, 0.5% sodium deoxycholate, and 1% Triton X-100), and then incubated with the post-ultracentrifugation lysates on a 4°C rotator overnight. Beads were then sequentially washed twice with 1 mL of normal RIPA buffer, once with 1 mL of 1 M KCl, once with 1 mL of 0.1 M Na₂CO₃, once with 1 mL of 2 M urea in 10 mM Tris-HCl [pH 8.0], and twice with 1 mL of normal RIPA buffer. For silver stain or Western blot, biointylated proteins were eluted by heating the beads at 95°C for 10 minutes in 20 μL of 3x protein loading buffer (Bio-Rad) supplemented with 20 mM dithiothreitol (DTT) and 2mM biotin. For proteomic samples, on-bead trypsin digestion was performed after enrichment (see below for details). All chemicals were purchased from Sigma-Aldrich.

Li J., Han S., Li H., Udeshi N.D., Svinkina T., Mani D.R., Xu C., Guajardo R., Xie Q., Li T., Luginbuhl D.J., Wu B., McLaughlin C.N., Xie A., Kaewsapsak P., Quake S.R., Carr S.A., Ting A.Y, & Luo L. (2020). Cell-Surface Proteomic Profiling in the Fly Brain Uncovers Wiring Regulators. Cell, 180(2), 373-386.e15.

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