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getting negative protein measurements


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#1 deejay

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Posted 21 November 2012 - 10:50 AM

Hello,

I've been having some trouble with my cell lysates in preparation for a western.
I collected my cells using RIPA buffer but after spinning them down to get rid of the nucleus, no pellet seems to show up.
We've had problems with our RIPA buffer before and finally got it fixed and this is the first time for me to use it ever since.

I went ahead and did the protein measurements using Bio-Rad assay but as expected, the measurements turned out to be very low or negative.

This is the second time in a row this has happened and I have no idea why the cells aren't getting lysed.

Any suggestions or help?

Thank you!

#2 bob1

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Posted 21 November 2012 - 11:36 AM

Check that the buffer components are compatible with bradford assay (assuming you are using the biorad bradford), this is especially important for the concentrations of detergents.

Make sure that the kit hasn't expired.

#3 Abhay_krishna

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Posted 22 November 2012 - 01:58 AM

Assuming you did biorad bradford:
Unlike other protein assays, the Bradford protein assay is less susceptible to interference by various chemicals that may be present in protein samples. An exception of note is elevated concentrations ofdetergent. Sodium dodecyl sulfate (SDS), a common detergent, may be found in protein extracts because it is used to lyse cells by disrupting the membrane lipid bilayer. While other detergents interfere with the assay at high concentration, the interference caused by SDS is of two different modes, and each occurs at a different concentration. When SDS concentrations are below critical micelle concentration (known as CMC, 0.00333%W/V to 0.0667%) in a Coomassie dye solution, the detergent tends to bind strongly with the protein, inhibiting the protein binding sites for the dye reagent. This can cause underestimations of protein concentration in solution. When SDS concentrations are above CMC, the detergent associates strongly with the green form of the Coomassie dye, causing the equilibrium to shift, thereby producing more of the blue form. This causes an increase in the absorbance at 595 nm independent of protein presence.

#4 Curtis

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Posted 22 November 2012 - 02:14 AM

I collected my cells using RIPA buffer


what do you mean you collected them with RIPA?

to add to bob1's comment I think your problem is lysis, not Bradford. First you must trypsinize or scrape your cells, centrifuge to pellet them down, then add 'cold' RIPA which has protease inhibitor. rotate on a rotator at 4C for a while (10 min enough, or you can just keep on ice but don't pipet up and down, it might give you a gooey DNA clump) and then centrifuge at high speed for 5 min. I must add that RIPA is a strong lysis buffer and breaks down all cell membrane including nuclei membrane. So the pellet at this stage is cellular debris including DNA, not intact nuclei. If you have collected a lot of cells then you must see a big pellet, if you didn't have a lot of cells then the pellet is smaller.

Also how much RIPA are you adding? for a T25 flask 100-1000 ul is enough.

I did Bradford on microplate, and I only got negative readings in my empty wells. my first standard sample was set zero.




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