4 replies to this topic

[Chiapet874]

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!

[mdfenko]

Chiapet874, on Jun 24 2010, 03:03 AM, said:

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?

Quote

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.

[Chiapet874]

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 >_>

[Chiapet874]

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

[mdfenko]

Chiapet874, on Jun 24 2010, 04:33 PM, said:

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.

Quote

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.