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protein sample degradation - (Dec/06/2010 )

Hi,

I'm working in a hospital and my topic involves use of pure compounds from Chinese medical herbs in sentisitizing effect of cancer cells to radiation. My supervising MD told me that his protein samples had higher degradation and his western blot results varied in samples (sets of samples collected at different days), so he suggested me to do protein quantification and western blot just right after samples are collected. But, in my experiences, WB results did vary from set to set and protease inhibitors (added in cell lysis buffer) decreased protein degradation, I don't know why my supervisors' samples degraded fast and why he thought doing WB after the day samples are collected was such a bad idea.




Rob

-Lee6948-

Hola, if your samples mantain more stability that your supervisor ones , dont worry but the supervisor is the supervisor... like he boss. Other alternative to run the WB days after, is made the quantification as soon as possible, take the necessary vol of sample for western, add loading buffer and frezze (boiled or not ) untill the moment of to do the PAGE. Other reason could be that your supervisor wants to have you busy every day that you have new samples. Good luck

-protolder-

So maybe it was due to certain technical problem that my supervisor created during the process?

-Lee6948-

Hola, probably yes, because Im thinking that you both use the same and the same concentration of primary antibody, if no , higher concentration of primary ab. and longer exposure times could lead to the apparition of more bands. Hasta pronto

-protolder-

Hi, yesterday I quantified my samples which were collected by using the same cell collecting method (the process took me 5 HR man and this was my first time using this method) provided by my supervisor and the protein concentration were around 2.2-4 ug/ul. This is enough for me to load 30 ug/well to western blot (2-3 gels), but I still think I should try my cell collecting method.

Here's his method:
1. Transfer 9 ml DMEM to 50-ml centrifuge tube, so all cells are collected (i.e. attached and floating)
2. Scrape cells down with the remaining 1 ml DMEM and transfer to the same tubes
3. Centrifuge for 10 min @ 800 rpm, 4C, and then discard DMEM
4. Add 1 ml PBS to each tube
5. Centrifuge for 10 min @ 800-2000 rpm, 4C
6. Discard PBS, add 70-80 μl RIPA cell lysis buffer (RIPA : protease inhibitor solution = 19:1 (v/v)) to each tube, rest tubes on ice for 30 min., mix by pipetting (for at least 10X) every 10 min and mix for few minutes before centrifusion
7. Centrifuge for 20 min @ 12000 rpm, 4C
8. Transfer supernatants to new tubes and store them @ -80℃


And this mine:

1. discard DMEM

2. was each plate with 3-4 ml PBS twice

3. eliminate remaining PBS

4. add 100 ul cell lysis buffer/plate and scraper down cells

5. transfer cells from one plate to an eppendorf tube

6. store samples @ -80℃ O/N

7. vortex each tube vigorously and place them on ice (3X)

8. centriguge for 20 min at 4℃

-Lee6948-

Hola, your method seems better, because washing cells in the plate eliminates floating cells that could be dead with the consequent DNA and protein degradation. Removing cells with lisys buffer is more efficient than with PBS and the cells go in few seconds from health to lisys avoiding degradation. To demostrate the efficiency of your method keep and analyze the cells of your first steps, floating in the medium and remove with the first wash, centrifuque add lisys buffer and compare with the extracted "in situ" with the lisys buffer. Buena suerte

-protolder-

Can you do cell viability before harvesting and lysis?

Would a general protein assay not provide overall concentration for monitoring?

It appears there is no 'control' protein to base this degredation observation..

-sgt4boston-

Huh

For the past 2 weeks, I finally got protein concentrations of 3-4 ug/ul because I made a few adustments to this new protein extraction method:

step 4: after sucking out DMEM, about 1 ml PBS was added to dissolve cell pellet (in each tube) by pipetting and the resulting mixture was transffered to a 1.5-ml microcentrifuge tube

step 5: centrifuge for 10 min @ 2000 rpm

step 6: carefuuly suck out PBS, added appropriate amount of RIPA (about 3X vol cell pellet), mix pellet with RIPA by tapping and vortexing and rest the 1.5-ml microcentrifuge tubes on ice for 10 min (3X), and finally vortex briefly before centrifusion

step 7: centrifuge for 30min @ 13000 rpm

-Lee6948-