Western blotting was performed on up to 20 μg of the cell lystes that were used for proteomic analysis (above). Proteins were separated by SDS-PAGE using a 4–15% Tris-HCl Ready Gel (Bio-Rad). The following primary antibodies were used at 1:1000 dilution in 5% milk, including: GAPDH (Millipore MAB374), RSPH4A (Sigma-Aldrich HPA031196), repetin (Sigma-Aldrich HPA030483), TGM1 (Sigma-Aldrich HPA040171), keratin 5 (Covance PRB160P), keratin 6 (Thermo PA5–28235), keratin 14 (Novus 34270) and keratin 17 (Cell Signaling 12509). The following secondary antibodies were used at 1:3000 dilution in 5% milk: goat anti-rabbit HRP (Santa Cruz Biotech sc-2004) and goat anti-mouse HRP (Santa Cruz Biotech sc-2005).
Keratin 14
Manufactured by Novus Biologicals
Sourced in United States
Keratin 14 is a type I intermediate filament protein expressed in basal cells of stratified squamous epithelia. It is a structural component of the cytoskeleton and is involved in maintaining the integrity and mechanical strength of epithelial cells.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti mouse hrp, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit hrp, by Santa Cruz Biotechnology (1 mentions)
Keratin17, by Cell Signaling Technology (1 mentions)
Tris hcl ready gel, by Bio-Rad (1 mentions)
Gapdh, by Merck Group (1 mentions)
Keratin 5, by Fortrea (1 mentions)
Cocktail of protease and phosphatase inhibitors, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Anti p53, by Santa Cruz Biotechnology (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
2 protocols using keratin 14
1
Western Blot Analysis of Cell Lysates
2
Protein Expression Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in RIPA buffer (150 mM NaCl, 1% SDS, 1% deoxycholic acid sodium salt, 0.1% sodium dodecyl sulfate, 50 mM Tris-HCl (pH 7.5), 2 mM EDTA; Thermo, Waltham, MA, USA) containing a cocktail of protease and phosphatase inhibitors (Thermo). Protein concentration was determined by Bradford assay (Bio-rad, Hercules, CA, USA). Total cell protein (30 μg) was electrophoresed on an SDS-polyacrylamide gel. Then, samples were transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Bedford, UK). Proteins of interest were detected by using following antibodies: anti-phospho Rb and keratin 14 (1:1,000 for phosphor Rb and 1:10,000 for keratin 14, NovusBio, CO, USA), anti-Rb (1:1,000 for Rb, Thermo), anti-p53 (1:1,000, Santa Cruz, CA, USA), anti-β-actin (1:10,000, Santa Cruz), horseradish peroxidaseconjugated anti-mouse, horseradish peroxidase-conjugated anti-Rabbit (1:10,000 for mouse and 1:20,000 for Rabbit; Thermo).
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!