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Protein Extraction from Tissue/ Lysate storage


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#1 Chiapet874

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Posted 23 June 2010 - 11:03 PM

Hello all!

Our lab has recently decided to do tissue protein analyses and I was tasked with finding out how. After doing a bunch of research across the web, I recognized that alot of lysis buffer recipes were for cell cultures. Is there any real reason or difference between a lysis buffer for tissue samples versus cell cultures?

For example, I plan on using a standard NP40-buffer provided by abcam:
150mM sodium chloride
1% Triton X-100
50 mM Tris, pH 8.0
+ some protease inhibitors courtesy of Pierce

Do you think this would work as a lysis buffer? I plan on using a homogenizing drill for my samples.

Also, it is fairly common knowledge that too many freeze/thaw cycles can ruin a protein lysate sample; however, just recently I read that mixing your lysate with 50% glycerol and stored at -20 degrees C can allow you to freeze/thaw your samples tons of times with no ill effect- any one else have any insight on the validity of this?

source: http://www.uni-leipz...age_Working.pdf

thanks again!

#2 mdfenko

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Posted 24 June 2010 - 07:06 AM

Our lab has recently decided to do tissue protein analyses and I was tasked with finding out how. After doing a bunch of research across the web, I recognized that a lot of lysis buffer recipes were for cell cultures. Is there any real reason or difference between a lysis buffer for tissue samples versus cell cultures?

For example, I plan on using a standard NP40-buffer provided by abcam:
150mM sodium chloride
1% Triton X-100
50 mM Tris, pH 8.0
+ some protease inhibitors courtesy of Pierce

Do you think this would work as a lysis buffer? I plan on using a homogenizing drill for my samples.

the triton should disrupt the cell membrane, so it should work. by the way, where is the np-40?


Also, it is fairly common knowledge that too many freeze/thaw cycles can ruin a protein lysate sample; however, just recently I read that mixing your lysate with 50% glycerol and stored at -20 degrees C can allow you to freeze/thaw your samples tons of times with no ill effect- any one else have any insight on the validity of this?

source: http://www.uni-leipz...age_Working.pdf

50% glycerol will prevent the solution from freezing at -20C, hence no freeze-thaw cycles.
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#3 Chiapet874

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Posted 24 June 2010 - 08:07 AM

ooo hmmm I wonder why I don't hear more people using the 50% glycerol in their lysates- probably because it will dilute their samples?

Also- I forgot to mention that I traded out the NP40 for triton x-100, abcam said it was alright >_>

#4 Chiapet874

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Posted 24 June 2010 - 12:33 PM

So today I did our first protein extraction from tissue and performed a bradford assay on the sample lysate.

Generally speaking, what is the "average" amount of protein concentration do you expect to find from brain tissue?

Using a bradford protocol with a nanodrop machine- I got around 6000 ug/ml... however, that is using BSA as a standard and I read that BSA usually underestimates concentrations by about 2-fold (bio-rad). So my "real" amount is 12,000 ug/ml... that seems like alot! O_o

To be fair though, there are protease inhibitors in the lysis buffer, but I don't know how to factor that out of my final protein concentrations.

thanks again! you are most helpful mdfenko

#5 mdfenko

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Posted 25 June 2010 - 07:13 AM

So today I did our first protein extraction from tissue and performed a bradford assay on the sample lysate.

Generally speaking, what is the "average" amount of protein concentration do you expect to find from brain tissue?

the "average" protein concentration and/or total protein extracted depends on the amount of starting material and volume of extraction. we used to homogenize ~1 kg gray matter (calf brain) in ~3 liter final volume. we obtained 16-20 gm of extracted proteins after centrifugation.

Using a bradford protocol with a nanodrop machine- I got around 6000 ug/ml... however, that is using BSA as a standard and I read that BSA usually underestimates concentrations by about 2-fold (bio-rad). So my "real" amount is 12,000 ug/ml... that seems like alot! O_o

To be fair though, there are protease inhibitors in the lysis buffer, but I don't know how to factor that out of my final protein concentrations.

you can run a subtractive buffer blank along with your samples to correct for the presence of inhibitors and other buffer components which may effect the readings.
talent does what it can
genius does what it must
i do what i get paid to do




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