Native sample buffer

Manufactured by Thermo Fisher Scientific

Sourced in United States

Native sample buffer is a solution used to prepare protein samples for analysis while maintaining their native structure and function. The buffer is designed to preserve the natural conformation and interactions of proteins during the sample preparation process.

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Sample buffer (138 citations) Tris-Glycine Native Sample Buffer (7 citations) Novex Tris-Glycine Native Sample Buffer (6 citations) Tris-Glycine SDS Native Sample Buffer (5 citations) Native gel sample buffer (5 citations) Native PAGE 4× Sample buffer (2 citations) Native gel electrophoresis sample buffer (2 citations)

14 protocols using native sample buffer

Measuring Complex I Activity in Mitochondria

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Complex I activity was measured in mitochondria isolated using Qproteome mitochondrial isolation kit (QIAGEN). 25 μg of mitochondrial protein was resuspended in 1x native sample buffer (Invitrogen), 2% digitonin (supercomplexes) or 2% DDM (n-dodecyl-β-D-maltoside) (isolated complex I), and 1x protease inhibitors (Roche) followed by 1 hour incubation on ice before final addition of 0.5% G-250 sample additive. Prepared samples were run on 3%–12% bis-tris native gels under native conditions at 150 V for 30 minutes in 1x native cathode buffer followed by 90 minutes at 150 V in 0.1x native cathode buffer. Following electrophoresis, gels were incubated in 150 μM NADH, 3mM nitro blue tetrazolium and 2 mM Tris at pH 7.4 for 3 hours.

Bailey J.D., Diotallevi M., Nicol T., McNeill E., Shaw A., Chuaiphichai S., Hale A., Starr A., Nandi M., Stylianou E., McShane H., Davis S., Fischer R., Kessler B.M., McCullagh J., Channon K.M, & Crabtree M.J. (2019). Nitric Oxide Modulates Metabolic Remodeling in Inflammatory Macrophages through TCA Cycle Regulation and Itaconate Accumulation. Cell Reports, 28(1), 218-230.e7.

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Analyzing Gamma-Secretase Complex

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Cells were suspended in a native sample buffer (Invitrogen) containing 1% digitonin and a protease inhibitor mixture. After centrifugation at 20,000 g, 4 °C for 30 minutes, the supernatant was separated onto the 4–16% Bis-Tris gel (Invitrogen) according to their molecular weight under the instructions of the Novex Bis-Tris gel system (Invitrogen). The γ-secretase complex levels were detected with a PS1-CTF antibody (Millipore).

Liu J., Liu S., Matsumoto Y., Murakami S., Sugakawa Y., Kami A., Tanabe C., Maeda T., Michikawa M., Komano H, & Zou K. (2015). Angiotensin type 1a receptor deficiency decreases amyloid β-protein generation and ameliorates brain amyloid pathology. Scientific Reports, 5, 12059.

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Native Mitochondrial Protein Complexes Analysis

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Isolated mitochondria (250 μg) of larvae tissues were suspended in BSA-free mitochondria isolation buffer (see above) and centrifuged at 10,000 × g at 4°C. Mitochondria (pellet) were resuspended in 100 μl 1X Native sample buffer (Invitrogen, BN20032) with either 1% Triton X-100 (Applichem, A4975) or 4% digitonin (Thermo Fischer Scientific, BN2006) and incubated for 10 min on ice before centrifuging at 20,000 × g at 4ºC for 30 min [80]. The supernatant (extracted solubilized complexes) was retained and Native PAGE sample additive G250 5% (Invitrogen, BN2004) was added. Samples were separated by 3–12% gradient BNGE and after electrophoresis the complexes were transferred on a polyvinylidene fluoride (PVDF) membrane and probed with the indicated antibodies.

Tsakiri E.N., Gumeni S., Vougas K., Pendin D., Papassideri I., Daga A., Gorgoulis V., Juhász G., Scorrano L, & Trougakos I.P. (2019). Proteasome dysfunction induces excessive proteome instability and loss of mitostasis that can be mitigated by enhancing mitochondrial fusion or autophagy. Autophagy, 15(10), 1757-1773.

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Assessing Mitochondrial Complex I Activity

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Fifty micrograms of isolated mitochondria were resuspended in 1× native sample buffer (Invitrogen) 2% digitonin and 1× protease inhibitors (Roche, Basel, Switzerland) and incubated for 1 h on ice before addition of 0.5% G-250 sample additive (Invitrogen). Prepared samples were run on NativePAGE™ 3–12% Bis-Tris gels under native conditions at 150 V for 30 min with 1× native cathode buffer (Invitrogen) followed by 90 min at 250 V in 0.1× native cathode buffer. The gel was then incubated for 1 h in 150 µM NADH, 3 mM nitro blue tetrazolium and 2 mM Tris-HCl (pH 7.4). Activity of Complex I corresponds to the depth of the blue-purple stain.

Nicol T., Falcone S., Blease A., Vikhe P., Civiletto G., Omairi S.S., Viscomi C., Patel K, & Potter P.K. (2023). Tissue-specific differences in the assembly of mitochondrial Complex I are revealed by a novel ENU mutation in ECSIT. Cardiovascular Research, 119(12), 2213-2229.

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Native 2D Gel Electrophoresis

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Cultured cells or brain microsomes were lysed in a lysis buffer containing 1% CHAPSO or 0.5% DDM. After preclearing lysates by ultracentrifugation at 100,000 g for 30 min, lysates mixed with native sample buffer (Thermo Fisher Scientific) and CBB G-250 (up to 1/4 of detergent concentration in each sample) were loaded on Native 3–12% Bis-Tris gel. After 150 min running at 150 V was done at 4°C, strips of gel were cut vertically and incubated with 50 mM DTT and 1% SDS for 2 h at 37°C. Presoaked strips were placed for the second dimension in Novex Bis-Tris gels with 2D wells, and electrophoresis was done using MES-SDS running buffer for an LMW target and MOPS-SDS running buffer for an HMW target.

Liu L., Ding L., Rovere M., Wolfe M.S, & Selkoe D.J. (2019). A cellular complex of BACE1 and γ-secretase sequentially generates Aβ from its full-length precursor. The Journal of Cell Biology, 218(2), 644-663.

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Native Protein Extraction and Separation

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HAP1 cells were prepared with a native sample buffer (Thermo Fisher Scientific), containing 0.5% DDM (n-dodecyl β-ᴅ-maltoside) and a protease inhibitor mixture and subjected to Blue Native PAGE, using the Novex Bis-Tris gel system (Thermo Fisher Scientific) in accordance with the manufacturer’s instructions.

Hara T., Maejima I., Akuzawa T., Hirai R., Kobayashi H., Tsukamoto S., Tsunoda M., Ono A., Yamakoshi S., Oikawa S, & Sato K. (2018). Rer1-mediated quality control system is required for neural stem cell maintenance during cerebral cortex development. PLoS Genetics, 14(9), e1007647.

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Quantifying Gamma-Secretase Complex

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HEK-APP cells or mouse cortices were homogenized in a native sample buffer (Thermo Fisher Scientific) containing 1% digitonin and a protease inhibitor mixture. After centrifugation at 20,000 × g at 4 °C for 30 min, the supernatant was separated on a 4–16% BisTris gel (Thermo Fisher Scientific) according to the instructions of the Novex BisTris gel system (Thermo Fisher Scientific). The γ-secretase complex levels were detected with PS1-CTF, APH-1, and NCT antibodies.

Noorani A.A., Yamashita H., Gao Y., Islam S., Sun Y., Nakamura T., Enomoto H., Zou K, & Michikawa M. (2021). High temperature promotes amyloid β-protein production and γ-secretase complex formation via Hsp90. The Journal of Biological Chemistry, 295(52), 18010-18022.

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Protein Extraction and Western Blotting

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Cells and tissue were homogenized in RIPA buffer (R0278, Sigma) with protease and phosphatase inhibitors (A32959, Thermo Scientific). Protein concentration was determined with a Pierce BCA Protein Assay Kit (23225, Thermo Scientific). Equal quantities of protein boiled in LDS Sample Buffer (NP0007, Thermo Scientific) were resolved by Novex Tris-glycine gels (Invitrogen) and transferred onto LF PVDF membranes (1704274, Bio-Rad).
For NATIVE PAGE, Cells were washed once with pre-chilled PBS and then lysed in ice-cold native lysis buffer (20 mM Tris-HCL, 20 mM NaCl, 10% (w/v) glycerol, 0.5% digitonin, 0.5 mM Na₃VO₄, 1 mM PMSF, 0.5 mM NaF, 1×Roche protease inhibitor cocktail, pH 7.5) for 30 min on ice. Cell lysates were centrifuged for 30 min at 12,000 rpm at 4 °C. Protein concentration was determined by Pierce BCA Protein Assay Kit. Equal quantities of protein were prepared in Native Sample Buffer (LC2673, Thermo Scientific) and resolved by 4–12% Native Tris-glycine gels. Native gels were incubated in 10% SDS solution for 5 min and transferred on to LF PVDF membranes. The transferred membranes were blocked with Blocking Buffer (927–40000, LI-COR) and incubated with the following primary antibodies. The signal was visualized with species-specific secondary antibodies conjugated with IRDye and Odyssey® CLx Imaging System.

Wang S.B., Narendran S., Hirahara S., Varshney A., Pereira F., Apicella I., Ambati M., Ambati V.L., Yerramothu P., Ambati K., Nagasaka Y., Argyle D., Huang P., Baker K.L., Marion K.M., Gupta K., Liu B., Hinton D.R., Canna S.W., Sallam T., Sadda S.R., Kerur N., Gelfand B.D, & Ambati J. (2021). DDX17 is an essential mediator of sterile NLRC4 inflammasome activation by retrotransposon RNAs. Science immunology, 6(66), eabi4493.

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Protein Extraction and Western Blot

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Cells were lysed in a native sample buffer (Thermo Fisher Scientific, USA) containing 1% digitonin and a protease inhibitor mixture. After centrifugation at 20,000 × g at 4 °C for 30 min, the supernatant was separated on a 3–12% BisTris gel (ThermoFisher Scientific, USA) according to the instructions of the Novex BisTris gel system (ThermoFisher Scientific, USA). The transferred blot was incubated in 20 mL of 8% acetic acid for 15 minutes to fix the proteins, rinsed with deionized water and analyzed with Western blotting.

Devkota S., Zhou R., Nagarajan V., Maesako M., Do H., Noorani A., Overmeyer C., Bhattarai S., Douglas J.T., Saraf A., Miao Y., Ackley B.D., Shi Y, & Wolfe M.S. (2024). Familial Alzheimer mutations stabilize synaptotoxic γ-Secretase-substrate complexes. Cell reports, 43(2), 113761.

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Non-denaturing Gradient Gel Electrophoresis of HDL

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Non-denaturing gradient-gel electrophoresis of HDL and FPLC fractions was performed as previously described [16 ]. Briefly, aliquots of HDL (10 μg) and FPLC fractions (15 μL) were electrophoresed on 4–16% non-denaturing polyacrylamide gels upon dilution with native sample buffer (LifeTechnologies, Vienna, Austria) at 125 V for 4 h at room temperature. Gels were stained with Sudan black (Sigma-Aldrich, Vienna, Austria) or fixed with 10% sulfosalicylic acid for 30 min and then stained with Coomassie Brillant Blue G250 or used for Western blotting analysis. The high molecular weight marker NativeMark (LifeTechnologies, Vienna, Austria) was used as standard.

Schilcher I., Ledinski G., Radulović S., Hallström S., Eichmann T., Madl T., Zhang F., Leitinger G., Kolb-Lenz D., Darnhofer B., Birner-Gruenberger R., Wadsack C., Kratky D., Marsche G., Frank S, & Cvirn G. (2019). Endothelial lipase increases antioxidative capacity of high-density lipoprotein. Biochimica et biophysica acta. Molecular and cell biology of lipids, 1864(10), 1363-1374.

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