Twenty to 80 μg of protein lysate was separated electrophoretically on denaturing SDS-polyacrylamide gel, transferred to nitrocellulose membranes, and probed with primary antibodies. Blots were exposed to horseradish peroxidase-conjugated secondary antibodies and visualized by an ECL detection system according to the manufacturer’s protocol (Amersham Biosciences, Piscataway, NJ). For loading control, the initial western blot was placed in Restore Western stripping buffer (Thermo Scientific) for 15 min to remove the antibody (primary and secondary), washed in water for 5 min, blocked with 5% milk for 1 hour, and probed with β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA; Catalog # sc-130301) [20 (link),24 (link)]. The primary antibodies used in western blot included NRP2, GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA. NPR2 catalog # sc-13117; GAPDH catalog # sc-137179); VEGF, DKK3 (Abcam, Cambridge, MA. VEGF catlog # ab46154; DKK3 catalog # ab136101); Wif-1 (Cell Signaling Technology, Danvers, MA, Catalog # 5652); Each western blot was repeated 3 times. Blots were quantitated by densitometry using Image J (Software, NIH, Bethesda, MD, USA) and normalized to a housekeeper marker β-actin or GAPDH [36 (link)]. The intensity of each tested marker is presented as a ratio of a tested mark/β-actin or GAPDH.
Restore western stripping buffer
Manufactured by Thermo Fisher Scientific
Restore Western stripping buffer is a laboratory reagent designed to remove primary and secondary antibodies from Western blot membranes, allowing them to be reused for subsequent detection of different target proteins. The buffer effectively strips away the bound antibodies, enabling the membrane to be reprobed with new antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions)
β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Vascular endothelial growth factor (vegf), by Cell Signaling Technology (1 mentions)
Ecl detection system, by Cytiva (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Abcam (1 mentions)
Mouse anti β actin antibody, by Cell Signaling Technology (1 mentions)
Sds polyacrylamide gel electrophoresis gel, by Bio-Rad (1 mentions)
Ecl plus, by GE Healthcare (1 mentions)
Goat anti rabbit igg horseradish peroxidase linked secondary antibody, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
4 protocols using restore western stripping buffer
1
Western Blot Analysis of Protein Markers
2
Immunoblotting Analysis of Murine T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
FACS-sorted murine Treg and Teff cells were lysed in lysis buffer [50 mM tris-Cl (pH 7.4), 150 mM NaCl, 2 mM EDTA, and 0.5% Triton X-100 including protease inhibitor cocktail]. Lysates were briefly sonicated, incubated for 30 min on ice, and precipitated at 12,000 rpm for 20 min. Twenty to 30 μg of the soluble fractions was run on 4 to 20% Bio-Rad SDS–polyacrylamide gel electrophoresis gels and transferred to polyvinylidene difluoride membranes. After blocking with 5% nonfat dry milk in tris-buffered saline containing 0.1% Tween 20 for 1 hour at room temperature, membranes were incubated with rabbit anti-PBX1 polyclonal antibody (Cell Signaling) or rabbit anti-RTKN antibody (Abcam) overnight at 4°C. Membranes were then washed and incubated with horseradish-conjugated IgG secondary antibody (Cell Signaling). Immunoblots were developed with Western enhanced chemiluminesence substrate (Cell Signaling). Bound antibodies were removed with the Restore Western Stripping Buffer (Thermo Fisher Scientific), and then membranes were incubated with mouse anti–β-actin antibody (Cell Signaling) to normalize protein content.
3
Protein-Protein Interaction Profiling
4
Western Blot Analysis of E-cadherin and α-SMA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NRK-52E cells were lysed in a lysis buffer (150 mM NaCl, 1 mM EDTA, 50 mM Tris, 1% NP-40, 1% Triton X-100, and 1% protease inhibitor cocktail (Sigma-Aldrich, Kyoto, Japan)) and centrifuged to collect the supernatant. The loading samples containing "1" to "5" μg of protein were electrophoresed on SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking the PVDF membranes with 5% nonfat milk in TBST (Tris-buffered saline (TBS) containing 0.1% Tween 20) for 1 h, the membranes were incubated with E-cadherin, α-SMA or GAPDH, diluted up to 1:2000, at 4°C overnight. Goat anti-rabbit IgG, horseradish peroxidase-linked secondary antibody (Cell Signaling Technology), diluted up to 1:2000 was detected by adding enhanced chemiluminescent reagent. Blots were stripped with Restore™ Western Stripping Buffer (Thermo Fisher Scientific) and re-probed with different antibodies. The band intensity was quantified from scanned membrane images using the ImageJ Fiji software.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!