Western blots were performed as described previously [20 (link),24 (link)]. Recombinant proteins or cell lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to polyvinylidene difluoride membranes (Merck Millipore, Burlington, MA, USA). After blocking with Blocking One solution (Nacalai Tesque, Tokyo, Japan), the membranes were incubated with primary antibodies, followed by incubation with respective horseradish peroxidase (HRP)-conjugated secondary antibodies. For the detection of α-DG, the lysates were enriched by wheat germ agglutinin (WGA)-agarose (Sigma-Aldrich) and subjected to Western blot analysis. The protein bands were developed with Immobilon Western Chemiluminescent HRP substrate solution (Millipore) and were imaged with an Amersham™ Imager 600 (GE Healthcare, Chicago, IL, USA). After removing the antibodies or avidin by Western Blot Stripping Buffer (Takara Bio Inc.), the membrane was reproved using different antibodies.
Western blot stripping buffer
Manufactured by Takara Bio
Sourced in Japan
Western Blot Stripping Buffer is a laboratory reagent used to remove primary and secondary antibodies from a Western blot membrane. This allows the membrane to be re-probed with different antibodies, enabling the analysis of multiple target proteins on the same sample.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon western chemiluminescent hrp substrate solution, by Merck Group (2 mentions)
Amersham imager 600, by GE Healthcare (2 mentions)
Blocking one solution, by Nacalai Tesque (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Wga agarose, by Merck Group (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Las 4000 mini system, by Fujifilm (1 mentions)
Immobilon fl polyvinylidene difluoride pvdf membrane, by Merck Group (1 mentions)
Rabbit anti stat3 antibody, by Cell Signaling Technology (1 mentions)
Odyssey clx, by LI COR (1 mentions)
Phosphatase inhibitor cocktail, by Nacalai Tesque (1 mentions)
Ripa buffer, by Bio-Rad (1 mentions)
Blocking one p, by Nacalai Tesque (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Hrp conjugated streptavidin, by Merck Group (1 mentions)
Anti his6 peroxidase, by Roche (1 mentions)
Amersham imager 600 analysis software, by GE Healthcare (1 mentions)
Anti mouse igg hrp antibody, by GE Healthcare (1 mentions)
5 protocols using western blot stripping buffer
1
Western Blot Analysis of α-DG
2
Western Blot Membrane Stripping and Reprobing
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Cell or tissue lysates and immunoprecipitates prepared as described above were resolved by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The proteins were then transferred to a nitrocellulose membrane that was blocked in Tris-buffered saline with 0.05% Tween 20 (TBST; pH 7.4) and 5% skim milk for 1 h at room temperature, followed by incubation with primary antibody in TBST with 10% foetal calf serum and 1% bovine serum albumin at 4 °C for 3 h or overnight. The membrane was washed three times in TBST and incubated with secondary antibody in TBST with 0.5% skim milk for 1 h at room temperature. After three washes in TBST, the membrane was developed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) and visualised on a LAS4000mini system (Fujifilm, Tokyo, Japan). The membrane was stripped by vigorous shaking in Western Blot Stripping Buffer (Takara Bio) at room temperature for 15–30 min, followed by three 10-min washes in TBST.
3
Western Blot Analysis of PAD4 and STAT3
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Lysates of the joints were prepared in TNE lysis buffer (10mM Tris, 150mM NaCl, 1mM EDTA with 1% Nonidet-P40) and the 20-µg equivalent whole cell crudes were boiled in sample buffer for 5 min, resolved on Sodium dodecyl sulfate-Poly-Acrylamide Gel Electrophoresis (SDS-PAGE), and transferred electrically onto Immobilon-FL PolyVinylidene DiFluoride (PVDF) membrane (Merck Millipore, Darmstadt, Germany). After blocking with fish gelatin, the membrane was incubated with rabbit anti-PAD4 antibody (Proteintech Group, Chicago, Illinois, USA), and rabbit anti-phospho-STAT3 (Tyr705) (pSTAT3) antibody (Cell Signaling Technology, Beverly, Massachusetts, USA), for overnight at 4°C. Bound antibodies were visualised with secondary antibody IRDye 800CW-labelled donkey anti-rabbit IgG (LI-COR, Lincoln, Nebraska, USA) with Odyssey CLx (LI-COR). Anti-pSTAT3 antibody was stripped with Western BLoT Stripping Buffer (TAKARA Bio, Shiga, Japan), and then the membrane was reprobed with rabbit anti-STAT3 antibody (Cell Signaling Technology).
4
Tacr2 Regulation of T Cell Activation Signaling
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CD8+ T cells (1 × 107) obtained from the spleen tissues of wild‐type or Tacr2−/− C57BL/6 mice were stimulated with anti‐CD3 and anti‐CD28 mAbs (50 ng/mL) for 20 min, and total and phosphorylated ERK, IκB, and α‐tubulin levels in cell lysates were detected by western blotting. Cells were lysed using RIPA buffer (Thermo) (Tris HCl 25 mM [pH 7.6], NaCl 150 mM, 1% sodium deoxycholate, 1% Nonidet P‐40, 0.1% SDS, protease inhibitor cocktail [Sigma‐Aldrich], and phosphatase inhibitor cocktail [Nacalai Tesque]). Proteins were then diluted in RIPA buffer and 3× Laemmli Sample Buffer (Bio‐Rad, Hercules, CA, USA), incubated at 95°C for 5 min, resolved by e‐PAGEL (E‐T1020L) gel electrophoresis (ATTO), transferred to a polyvinylidene difluoride membrane, and blocked in Blocking ONE‐P (Nacalai Tesque) for 1 h at room temperature. Membranes were then incubated with antibodies (phospho‐ERK 1:1000, IκBα 1:1000, α‐tubulin 1:1000) at 4°C overnight, followed by HRP‐conjugated anti‐rabbit IgG (1:5000) or HRP‐conjugated anti‐mouse IgG (1:5000) secondary antibody at room temperature for 1 h. The protein bands were visualized using an ImageQuant™ LAS 4000 mini system (Bio‐Sciences) and ImageQuant™ LAS 4000 software (Bio‐Sciences) and analyzed and quantified using ImageJ software. The membranes were reprobed after removing bound antibodies using Western Blot Stripping Buffer (TaKaRa).
5
Immunoblot Analysis of Recombinant Proteins
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Immunoblot analysis was performed as previously described10 (link). Recombinant proteins or cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). After blocking with Blocking One solution (Nacalai Tesque), the membranes were incubated with monoclonal mouse anti-Flag M2 antibody (Sigma-Aldrich, F1804) and AK9733 , followed by incubation with respective horseradish peroxidase (HRP)-conjugated secondary antibodies, anti-mouse IgG antibody-HRP (GE Healthcare) and anti-mouse IgM antibody-HRP (ENZO), respectively. The hexahistidine (His6)-tagged and biotin-labeled recombinant proteins were detected using anti-His6-Peroxidase (Roche) and HRP-conjugated Streptavidin (Sigma), respectively. The protein bands were developed with Immobilon Western Chemiluminescent HRP substrate solution (Millipore) and were imaged with an Amersham™ Imager 600 (GE Healthcare). After removing the antibodies or avidin by Western Blot Stripping Buffer (Takara Bio Inc.), the membrane was reproved using different antibodies. For deglycosylation, lysates were incubated with 50 units/mL of PNGase F (New England BioLabs) at 37 °C for 48 h before being subjected to SDS-PAGE. Densitometry analysis was performed using Amersham™ Imager 600 Analysis Software (GE Healthcare).
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