Application
Western blot
Sample
Human Cell lysate - whole cell (primary keratinocytes)
Gel Running Conditions
Reduced Denaturing (any kD BioRad)
Loading amount
50 µg
Specification
primary keratinocytes
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Other product details
Incubation time
12 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: TBS-T 0.1%
Dilution
1/1000
Secondary antibody
Dilution
1/8000
Name
Non-Abcam antibody was used: goat anti-rabbit HRP
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Detection
Detection method
ECL
Exposure
5 second(s)
Bands
Specific: 76 ? kDa Non-specific: 48 kDa
Additional data
Additional Notes
I am not sure about the real molecular weight of this protein
Abcam response
The predicted band size for Cytokeratin 1 is 66 kDa (SwissProt# P04264).
The "predicted" band size is based entirely on amino acid sequence (or the size of the protein). However, there are other factors which may play a part in determining the observed size of the protein in an actual western blot.
1. Protein modification: Post translational cleavage where a larger pro-form of the protein is cleaved into a smaller active form which decreases its size in the gel or post-translational modification where a protein becomes glycosylated (N or O linked sites), phosphorylated or ubiquitinated increasing its size in the gel.
2. The overall net protein charge determined by the amino acid composition may affect the migration speed through the negatively charged SDS of the gel.
3. Trimerization, or dimerization creating multimeric proteins. The use of reducing conditions will help to eliminate these interactions.
4. Alternative splicing may produce differently sized proteins from the same ge
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
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投稿 Jul 02 2018