To evaluate knockdown of SMAD6, HUVEC whole-cell lysates were prepared in RIPA buffer supplemented with protease/phosphatase inhibitor cocktail (Cell Signaling 5872S). Lysates were separated on 10% TGX Stain-Free FastCast SDS–PAGE gels (Bio-Rad 161–0183), followed by 1 min ultraviolet activation of TGX stain for total protein quantification. Protein was transferred to polyvinylidene difluoride membranes (Bio-Rad 162–0177), blots were imaged under ultraviolet for total protein, blocked for 1 hr in 5% non-fat milk (NFM) in PBS+0.1% Tween20 (PBST), then incubated overnight at 4°C with SMAD6 primary antibody (1:5,000, Abcam ab13727) or GAPDH (1:15,000, Cell Signaling 97166S) in 1% NFM in PBST. After washing, horseradish peroxidase-conjugated secondary antibodies (1:15,000, Life Technologies G21234 or 816720) were added for 1 hr at RT in 1% NFM in PBST. Luminata Forte (Millipore WBLUF0100) was used for detection. Blots were images on a Bio-Rad ChemiDoc XRS system and band densities were calculated on Bio-Rad Image Lab software, following established guidelines [55 (link)].
Tgx stain free fastcast sds page gels
Manufactured by Bio-Rad
10% TGX Stain-Free FastCast SDS–PAGE gels are pre-cast polyacrylamide gels used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation of proteins. The gels are formulated with a proprietary stain-free technology that allows for direct visualization of proteins without the need for traditional staining methods.
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Lab products found in correlation
Polyvinylidene difluoride membrane, by Bio-Rad (2 mentions)
G 21234, by Thermo Fisher Scientific (1 mentions)
Luminata forte, by Merck Group (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Clarity ecl, by Bio-Rad (1 mentions)
Protease phosphatase inhibitor cocktail, by Cell Signaling Technology (1 mentions)
2 protocols using tgx stain free fastcast sds page gels
1
SMAD6 Knockdown in HUVEC Cells
2
Western Blot Analysis of HUVEC Lysates
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HUVEC whole-cell lysates were prepared in RIPA buffer supplemented with protease/phosphatase inhibitor cocktail (Cell Signaling 5872S). Ten micrograms of whole-cell lysates were separated on 10% TGX Stain-Free FastCast SDS–PAGE gels (Bio-Rad 161–0183), followed by 2 min ultraviolet activation of TGX stain for total protein quantification. Proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad 162–0177), blots were imaged under ultraviolet for total protein, blocked for 2 h in 5% non-fat milk (NFM) in PBS+0.1% Tween20 (PBST), then incubated overnight at 4 °C with primary antibody in 1% NFM in PBST (see Supplementary Table 1 for primary and secondary antibodies and concentrations). Horseradish peroxidase-conjugated secondary antibodies (Life Technologies goat anti-rabbit G21234 or rabbit anti-mouse 816720) were added for 2 h at RT in 1% NFM in PBST. Clarity ECL (Bio-Rad 170–5060) was used for detection. Films were digitized using an AlphaImager (ProteinSimple) gel scanning station and band densities calculated in FIJI52 (link), following established guidelines53 (link).
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