I couldn't detect Vimentin in western blot, in spite of positive result in qPCR and Immuno Fluorescence. Any suggestions?

[dvimal27]

I work on Epithelial to Mesenchymal Transition (EMT) using MDA-MB-468 as a model cell line. I use EGF as an EMT inducing signal. I’ve done imaging to confirm that cells undergo EMT post EGF treatment. They become more elongated and spindle. I’ve also done qPCR that shows 20 fold higher vimentin expression upon EGF treatment. I’ve also done Immuno fluorescence with anti vimentin (ab195877 – abcam), which shows higher amount of vimnetin in EGF treated cells. But I am unable to detect vimentin in western blot. However I have a positive control lane in the SAME BLOT (cell lystae from MDA-MB-231 – vimentin positive cell line), that gives a prominent band at the expected size. This confirms that there are no issues with antibody, gel and transfer. I use primary antibody from abcam (ab92547). What could be the probable reason?

 

I tried the following:

· Loaded 80 ug of total protein

· I tried with many cell lysis buffer (RIPA buffer, Tris-triton buffer, Nuclear extraction buffer, whole cell lysis buffer) . All these buffers contains protease inhibitor cocktail from SIGMA (P8340) + sodium ortho vandate + sodium fluoride + PMSF

 

This is how I prepare cell lysate:

· Remove media from plate (35 mm petri dish)

· Wash with PBS

· Add 100 uL of cell lysis buffer. Keep it on ice for 10 min

· Scrap the cells. Collect the cells, keep it on ice for 20 min.

· Centrifuge it at 12,000 rpm for 10 min

· Store the supernatant (cell lysate) at -80

Edited July 18, 2017 by dvimal27

[CharonY]

I assume you have measured protein concentration after lysis to ensure quantitative release of proteins? Have you tried a positive control on your sample (e.g. GAPDH?) to ensure that nothing is wrong with it?

qPCR is a relatively weak evidence of presence as the typical semi-quantiative nature of the measurement does not necessarily tell you much about detectability. Though a positive immuno-stain (assuming it was specific) is usually a good indicator.

Edited July 18, 2017 by CharonY

[dvimal27]

I assume you have measured protein concentration after lysis to ensure quantitative release of proteins? Have you tried a positive control on your sample (e.g. GAPDH?) to ensure that nothing is wrong with it?

qPCR is a relatively weak evidence of presence as the typical semi-quantiative nature of the measurement does not necessarily tell you much about detectability. Though a positive immuno-stain (assuming it was specific) is usually a good indicator.

I could detect other proteins like E-cadherin, beta tubulin with the same sample. They are completely fine with prominent band at the expected size. Is there any possibility of protein (Vimentin) being in insoluble fraction (pellet that is discarded)?

[CharonY]

That is odd. It is possible, that they may not have gone into solution, but typically the strong detergents in the lysis buffer should take care of it. Unless you have a formulation with only mild buffers (e.g. NP-40, Triton) in which case I would add SDS.