I recently did the baseline levels of some proteins in different tumor cell lines and used b-actin as equal loading control. I am struggling to get equal b-actin in different cell types. I have encountered this problem in the past also. However, I almost always get equal b-actin when the samples are derived from same cell type with different experimental conditions. I am thinking that b-actin levels might be variable in different tumor cells? If this is the case, could someone suggest me better equal loading control for this type of experiments?
Thanks
You cannot compare any protein across different cell lines. There is nothing that will give you what you want.
I aggree with Terrance,
Structural Proteins like actin or tubulin depend on size and shape of a cell type. Metabolic proteins like GAPDH depend on the general metabolic status of cells. HSC70 will depend on generel protein folding capacity of the cell type.
You can adjust the protein expression to cell number (very exact counting is needed) but there is no internal loading control.
It depends but one of the most consistent protein across cell lines is Histone H3. You can try GAPDH or TBP as well. Better to check all of these if you have the antibodies.
I am using GAPDH as a loading control in A549 (lung cancer), MDA-MB-231 and MCF7 (breast cancer), SHSY5Y (neuroblastoma) and HCT116 (colon cancer). The bands are quite similar in each cell line.
GAPDH or Tubulin. Might be an good alternative for equal loading protein marker.
Hi Sajid,
Instead of using the single loading control across the different cell lines, You should go for total protein normalization using the stain-free gel.
Hi Sajid,
you might want to check out the following paper for selecting a loading control: Article Comparative Proteomic Analysis of Eleven Common Cell Lines R...
Here the authors assessed protein expression levels across 11 different cancer cell lines using mass-spectrometry. For example, they found that ribosomal proteins are in general expressed at the same level for all cell lines. One of them may be thus be suitable as loading control in your experiments.
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