Unable to detect Vimentin in western blot. Any suggestions?

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Unable to detect Vimentin in western blot. Any suggestions?

Postby dvimal27 » Jul 18 2017 6:00 am

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 (15 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
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Re: Unable to detect Vimentin in western blot. Any suggestio

Postby relaxin » Jul 19 2017 4:41 pm

It looks like either the expression of vimentin is too low for detection or you have not extracted it from the cells. Most of the vimentin may remain insoluble. You can boil the cell pellet with 4X Laemmli sample buffer, sonicate for 15 sec, and then centrifuge.

I notice that you do not sonicate your cells to break down the DNA, you may lose a lot of proteins trapped in the DNA.
Retired academic researcher. Mention of a specific product does not imply my endorsement of the product. No conflict of interest or guarantee to work on the advice given. Do as I say, not as I do. Not liable to the loss of your valuable samples.
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Re: Unable to detect Vimentin in western blot. Any suggestio

Postby dvimal27 » Jul 21 2017 4:58 am

relaxin wrote:It looks like either the expression of vimentin is too low for detection or you have not extracted it from the cells. Most of the vimentin may remain insoluble. You can boil the cell pellet with 4X Laemmli sample buffer, sonicate for 15 sec, and then centrifuge.

I notice that you do not sonicate your cells to break down the DNA, you may lose a lot of proteins trapped in the DNA.



Thank you for your suggestion. Definitely I would try that and let you know
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