Respected Members,
I am performing western blot on A549 cells after transfecting with siRNA silencing a particular gene that plays a role in antioxidant response. After exposing the siRNA-treated cells with cigarette smoke extract i am not getting consistent beta-actin and GAPDH signal as loading controls between smoke-treated and smoke-untreated groups.
N.B. I am loading 30 ug of protein to each lane. But the LC signal significantly varies between treated and untreated groups. I have checked for cytotoxicity for transfection but nothing seems to be wrong. In such a scenario I am unable to find a suitable loading controls for my western blot experiments. How can I use total protein as a internal loading control?
Please email me an image of your problem at [email protected] (or attach an image to your previous post).
I suggest you take a look at the article below:
Rocha-Martins M, Njaine B, Silveira MS. Avoiding pitfalls of internal controls: validation of reference genes for analysis by qRT-PCR and Western blot throughout rat retinal development. PLoS One. 2012;7(8):e43028.
It seems that the loading controls are not appropriate and your compound in question is affecting them. you should try using other controls like beta tubulin , 18sRNA etc. if you are looking any signalling proteins probably non phosphorylated forms could be used. you can also try using ang survival kinase related proteins non phospho ERK,AKT etc as loading controls.
Having GAPDH changed is not surprising as you are affecting the metapolitic potential with your antioxidant modulation.
Actin structure and amount might change in cells during differentiation so it worth to check if your treatment is causing differentiation. And the same apply to vinculin.
Give a try to Tubulin or find a transcription factor that is not affected by your treatment.
You also might think about to use a simple Ponceau Red S stain of the whole membrane.
See here more:
https://www.researchgate.net/post/Can_Ponceau_staining_of_membranes_be_used_as_a_loading_control
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