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Restore plus stripping buffer

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Restore Plus stripping buffer is a laboratory reagent designed to remove bound antibodies or proteins from membranes or blots. It is a versatile solution that can be used to prepare samples for subsequent immunodetection or analysis.

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Lab products found in correlation

Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (5 mentions) Super signal west pico chemiluminescent substrate ecl, by Thermo Fisher Scientific (2 mentions) Nupage bis tris gel, by Thermo Fisher Scientific (2 mentions) Ecl reagent, by Merck Group (2 mentions) Image lab software, by Bio-Rad (2 mentions) Dc protein assay, by Bio-Rad (2 mentions) Protease and phosphatase inhibitor, by Thermo Fisher Scientific (2 mentions) Bradford reagent, by Bio-Rad (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Protease and phosphatase inhibitor, by Merck Group (2 mentions) Ripa buffer, by Thermo Fisher Scientific (2 mentions) Acetylated lysine, by Cell Signaling Technology (1 mentions) Anti acetylated lysine antibody, by Cell Signaling Technology (1 mentions) Protein a g plus agarose bead, by Santa Cruz Biotechnology (1 mentions) Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions) Protease inhibitor cocktail, by Santa Cruz Biotechnology (1 mentions) Stat6, by Cell Signaling Technology (1 mentions) P stat6, by Cell Signaling Technology (1 mentions) Image studio lite software, by LI COR (1 mentions) Laemmli buffer, by Bio-Rad (1 mentions)

33 protocols using restore plus stripping buffer

1

Multifaceted Analysis of Protein Acetylation

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Proteins were extracted with RIPA buffer (50 mM Tris-HCL [pH 7.5], 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with NaF, NaVO4, and Protease Inhibitor Cocktail (Cat.#P2714). Proteins were then separated in SDS-PAGE and were transferred to a nitrocellulose membrane, which was blotted with antibodies against Parkin (Cat.#2132), LC3 (Cat.#.2775), acetylated lysine (Cat#.9441) (all from Cell Signaling), cyclophyllin D (CypD, sc-137216; Santa Cruz), or Erk (SC-93; Santa Cruz). Protein bands were visualized using horseradish peroxidase-conjugated secondary antibodies and Supersignal West Femto Maximum Sensitivity Substrate (Cat.#34095; ThermoFisher). To determine acetylation levels of CypD, equal amounts of cell lysates were incubated with anti-CypD antibody overnight at 4°C, and were further incubated with protein A/G plus-agarose beads (sc-2003; Santa Cruz) for 2 h at 4°C. After washing, beads were precipitated, and proteins were processed for western blot analysis using anti-acetylated-lysine antibody (Cat.#9441; Cell Signaling). To visualize levels of CypD in the blot, the filter was stripped off the anti-acetylated-lysine antibody using Restore™ Plus stripping buffer (Cat.#46430; Thermo Scientific), and was then probed with anti-CypD antibody.
Song S.B., Jang S.Y., Kang H.T., Wei B., Jeoun U.W., Yoon G.S, & Hwang E.S. (2017). Modulation of Mitochondrial Membrane Potential and ROS Generation by Nicotinamide in a Manner Independent of SIRT1 and Mitophagy. Molecules and Cells, 40(7), 503-514.
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2

Western Blot Analysis of Protein Expression

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Treated HUVECs were washed with cold PBS and lysed in RIPA lysis buffer containing 1X phosphatase and protease inhibitor cocktail and centrifuged to obtain cell supernatants. Heart and aorta tissues were homogenized in RIPA buffer containing a protease inhibitor cocktail (Chem Cruz, Santa Cruz Biotechnology) with a tissue homogenizer. The tissue homogenates were centrifuged at 10,000 g for 20 min at 4°C. Total protein in the cell extracts and tissue homogenates was measured by using Bradford reagent (Bio-Rad protein assay, Bio-Rad). Equal amounts of proteins from cell lysates and tissue homogenates were subjected to SDS-PAGE followed by transfer of proteins to nitrocellulose membranes and probing with the specific antibodies. The antigen-antibody complexes were detected by enhanced Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific). All blots were re-probed with GAPDH as loading control after stripping with Restore plus stripping buffer from Thermo Fisher Scientific. Densitometric analysis of western blots was performed using Image J.
Pal P.B., Sonowal H., Shukla K., Srivastava S.K, & Ramana K.V. (2019). Aldose reductase regulates hyperglycemia-induced HUVEC death via SIRT1/AMPK-α1/mTOR pathway.. Journal of molecular endocrinology, 63(1), 11-25.
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3

Western Blot Protein Analysis

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Equal amounts of cell lysates (40 μg) were loaded and separated on 12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were then blocked with 5% nonfat dried milk and incubated with the specific primary antibodies at 4°C overnight followed by incubating with the specific secondary antibodies. Immunolabeling was detected using SuperSignal™ West Pico Chemiluminescent Substrate (ECL) from Thermo Scientific following manufacturer's instructions. The membranes were stripped with Restore™ PLUS stripping buffer from Thermo Scientific following manufacturer's instruction for reprobing with other antibodies or GAPDH.
Sonowal H., Pal P.B., Shukla K, & Ramana K.V. (2017). Aspalatone Prevents VEGF-Induced Lipid Peroxidation, Migration, Tube Formation, and Dysfunction of Human Aortic Endothelial Cells. Oxidative Medicine and Cellular Longevity, 2017, 2769347.
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4

Western Blot Analysis of HUVECs

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Growth-arrested HUVECs were stimulated with HG (25 mM) in the absence and presence of didymin (20μM) at different time points. Cells were lysed with RIPA buffer and protein extract was collected. Equal amounts of protein in the cell lysates were separated on 12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were then blocked with 5% nonfat dried milk and incubated with the specific primary antibodies at 4°C overnight followed by incubating with the specific secondary antibodies. Immunolabeling was detected using SuperSignal West Pico Chemiluminescent Substrate (ECL) from Thermo Scientific (Waltham, MA, USA). The membranes were stripped with Restore PLUS stripping buffer from Thermo Scientific following manufacturer’s instructions and reprobed with other antibodies.
Shukla K., Sonowal H., Saxena A, & Ramana K.V. (2018). Didymin prevents hyperglycemia-induced human umbilical endothelial cells dysfunction and death. Biochemical pharmacology, 152, 1-10.
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5

Western Blot Analysis of Signaling Proteins

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Cells were washed with 1X PBS and resuspended in RIPA buffer with Halt protease inhibitor cocktail (Thermo Scientific). Lysates were centrifuged at 10,000 g for 10 min at 4°C, resuspended in Laemmli buffer (Bio-Rad) and β-mercaptoethanol, and boiled for 5 min. Protein was quantified using the BCA protein assay (Thermo Scientific Pierce) and 35 μg was run on SDS-PAGE gels (Bio-Rad) in 1X Tris-glycine-SDS buffer (Bio-Rad) at 120 V, and transferred using 1X Tris-glycine onto a.2 μM PVDF membrane for 1 h at 100 V. Membranes were blocked in 5% BSA (phospho antibodies) or 5% non-fat milk (other antibodies) with TBS-T. Membranes were incubated with primary antibody (pSTAT3, STAT3, pSTAT, STAT1, pSTAT6, STAT6 (Cell Signaling Technology, 1:1000), AID (Active Motif, 1:500), Actin (Sigma, 1:10,000)) and in secondary antibody (Anti-rabbit, anti-mouse, goat-anti-rat; 1:10,000) and developed with Super Signal West Pico ECL western blotting substrate (Pierce) per the manufacturer’s instructions. Membranes were stripped with Restore PLUS Stripping Buffer (Thermo Fisher Scientific) per manufacturer’s instructions. Images were quantified by densitometry with Image Studio Lite software (Li-cor).
Rosario S.A., Santiago G.E., Mesri E.A, & Verdun R.E. (2018). Kaposi’s Sarcoma-Associated Herpesvirus-Encoded Viral IL-6 (vIL-6) Enhances Immunoglobulin Class-Switch Recombination. Frontiers in Microbiology, 9, 3119.
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6

Western Blot Protein Analysis

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After the indicated treatments, the cells were collected using a scrapper, centrifuged, washed with ice-cold PBS, and lysed with RIPA buffer containing phosphatase and protease inhibitors. Equal amounts of cell lysates were loaded and separated by 12% SDS-PAGE, followed by the transfer of proteins on to PVDF membranes. The membranes were then blocked with 5% non-fat dried milk and incubated with the specific antibodies overnight. The next day, the membranes were washed, incubated with specific secondary antibodies. Immunolabeling was detected using a SuperSignal West Pico chemiluminescent substrate (#34078, Thermo Scientific). For re-probing with different antibodies or loading control, the membranes were stripped with Restore Plus stripping buffer (#46430, Thermo Scientific).
Sonowal H, & Ramana K.V. (2020). 2’-Hydroxyflavanone prevents LPS-induced inflammatory response and cytotoxicity in murine macrophages. Toxicology in vitro : an international journal published in association with BIBRA, 69, 104966.
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7

Western Blot Protein Analysis Protocol

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Cells were washed with PBS and lysed in nonreducing Laemmli buffer (50mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol). Lysates were boiled for 5 minutes to shear DNA. Total protein was quantified by bicinchoninic acid (BCA) assay (ThermoFisher), then lysates were supplemented with β-mercaptoethanol (final concentration 200mM) and boiled for 5 minutes. Equal amounts of total protein (typically 30 μg) were separated on 4%-12% NuPAGE Bis-Tris gels (Invitrogen) in MOPS SDS buffer and transferred to a PVDF membrane (Pierce, 88518) using liquid transfer in Tris-Glycine buffer supplemented with 10% methanol. Membranes were blocked in 5% skim milk dissolved in PBS-T (PBS plus 0.05% Tween-20) at room temperature for 1h. All antibody incubations and washes were performed in PBS-T at room temperature. Protein bands were detected using standard chemiluminescence techniques. Blots were stripped in Restore™ PLUS Stripping Buffer (ThermoFisher, 46430) and re-probed with multiple antibodies.
Addison J.B., Voronkova M.A., Fugett J.H., Lin C.C., Linville N.C., Trinh B., Livengood R.H., Smolkin M.B., Schaller M.D., Ruppert J.M., Pugacheva E.N., Creighton C.J, & Ivanov A.V. (2021). Functional hierarchy and cooperation of EMT master transcription factors in breast cancer metastasis. Molecular cancer research : MCR, 19(5), 784-798.
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8

Western Blot Analysis of Cellular Proteins

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Cell were treated as for qPCR experiments. To prepare protein lysates, cells were harvested, washed, and lysed in ice cold RIPA buffer (150 mM NaCl, 5 mM Tris [pH 8.0], 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with complete protease inhibitor cocktail (Roche) and PhosSTOP phosphatase inhibitors (Roche). Western blotting was performed according to standard protocols. Specific antibodies against AHR (NB100-2289, Novus Biologicals) MDM2 (33–7100, ThermoFisher Scientific), p21 (610233, BD Transduction), p53 (sc-126, Santa Cruz Biotechnology), p53 p-Ser33 (2526 s, Cell Signaling), p53 p-Ser15 (9286 S, Cell Signaling), p53 p-Ser37 (9289 S, Cell Signaling) were used for endogenous protein detection. As loading control, anti-β-actin (MAB1501, Millipore) or anti-GAPDH (sc-365062, Santa Cruz Biotechnology) was used after stripping the membrane with Restore Plus Stripping Buffer (ThermoFisher Scientific). Densitometric analysis of the bands was performed using ImageJ software (http://imagej.nih.gov/ij/). Full-length uncropped blots are displayed in Supplementary Fig. S4.
Zhu J., Singh M., Selivanova G, & Peuget S. (2020). Pifithrin-α alters p53 post-translational modifications pattern and differentially inhibits p53 target genes. Scientific Reports, 10, 1049.
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9

Curcumin and Glucuronide Assays for Inflammatory Pathways

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Curcumin (218580100, Fisher; 80.6% curcumin, 13.5% demethoxycurcumin, and 2.4% bisdemethoxycurcumin by weight) and curcuminglucuronide were purchased, with content/purity verified using standard LC/MS methods (see below)11 (link),60 (link) with stock solutions prepared in DMSO. Murine RAW 264.7 cells were obtained from American Type Culture Collection (#TIB-71), ATCC) and used within 10 passages. Primary antibodies against GUSB (ARP44234_T100, Aviva Systems Biology), HPSE (bs-1541R, Bioss USA), KL (ab181373, Abcam) and HRP secondary antibodies (7074, Cell Signaling) were purchased. Phenolphthalein-glucuronide (PhePG; P0501), 4-methylumbelliferyl-glucuronide (4-MUG; M9130), D-Saccharic acid 1,4-lactone monohydrate (saccharolactone, S0375), and RIPA lysis buffer (R0278) were purchased from Sigma Aldrich. Supersignal West Femto ECL (34095) and RestorePlus stripping buffer (46430) were purchased from ThermoFisher. Recombinant human (rh)-GUSB (6144-GH), rh-HPSE (7570-GH), and rh-KL (5334-KL), and recombinant mouse receptor activator of NFkB ligand (RANKL, 462-TEC) were purchased from R&D Systems. Mini-PROTEAN TGX-PAGE gels (4568046) were purchased from BioRad and PVDF membranes (IPFL0010) from Millipore.
Kunihiro A.G., Luis P.B., Brickey J.A., Frye J.B., Chow H.H., Schneider C, & Funk J.L. (2019). Beta-glucuronidase catalyzes deconjugation and activation of curcumin-glucuronide in bone. Journal of natural products, 82(3), 500-509.
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10

Western Blot Analysis of Proteins

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Equal amounts of nuclear and cytoplasmic proteins were subjected to Western blot analysis using specific antibodies. The antigen-antibody complexes were detected by enhanced Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific). Membranes were stripped with Restore™ PLUS stripping buffer (Thermo Scientific) and used for reprobing with other antibodies or loading control. Densitometric analysis was performed using Image J software (NIH) and fold changes were calculated by normalizing to either loading control GAPDH or respective total proteins.
Shukla K., Sonowal H., Saxena A., Ramana K.V, & Srivastava S.K. (2017). Aldose reductase inhibitor, fidarestat regulates mitochondrial biogenesis via Nrf2/HO1/AMPK pathway in colon cancer cells. Cancer letters, 411, 57-63.
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