Tai

Thank you for your response!

bob1, on 08 May 2013 - 01:57 PM, said:

My question is about when should I design to run WB with the native protein?
and what is the different of the result of the native protein or the denatured protein from WB ?
or is there any meaning to interpret our results, if we use the native protein or the denatured protein?

mdfenko, on 09 May 2013 - 04:54 AM, said:

That mean, it depends on antibody that is available for our particular protein, right ?
Then, we can design to prepare the lysis buffer for native or denature sample.

Try to answer what people ask on this forum ....

Teach students and you will get a lot of questions to find out

Thank you very much for your reply as always !!

Normally, I made stock Laemmi buffer and kept them in-20 C, I just need to have the same batch of laemmli buffer because I run experiment for the whole year to collect my cell samples.

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when I need to mix with my cell sample, I will add fresh of these inhibitors.

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So far it always work fine with other cells but I just got the new cells line, MCF10A, and its total protein concentration is about 4-6 time higher than other cell line that I have. So, I have to add more laemmli buffer than other cells lines

Other cell line 1x10^6 cells per 300 ul Laemmli
This MCF10A 1x10^6 cells per 600 ul Laemmli <------ Is this could be to make much more salt content in my sample?

If the salt content in the sample could make the "spot" problem -- Do you mean it was from cells content or from the laemmli buffer?

From the actin.jpg, if look at the first two lane.

Lane 1. total protein concentration 1.02 ug/ul and I loaded 7.3 ul to get 7.5ug
Lane 2. total protein concentration 0.39 ug/ul and I loaded 19.3 ul to get 7.5ug
Lane 3 is repeated of lane 1.

Other lane also about 0.4 ug/ul of total protein.

PS. I just bought new gel, and it is about 1 month from company

Hi
Thank you so much for clearifying with this images

After 2 month that I have tried with this HPRT Forward mutation assay, finally I could get colony. Wooohoooo!!

Here is my briefly protocol for Fibroblast, in case someone need!

First, Checking Killing curve with 6-TG some cell very sensitive some doesn't (try between 2-20 ug/mL)
Secondly, find the good positive control. ( I use UVC 0,48 j/m^2 and irradiated for 20sec to induce mutation)

1. Stock cells about 80% confluent, trysinizing and count number of cells about 3x10^6 cells
2. wash cell twice with HBSS without phenol red ( red color can absorb UVC light) in petridish 100mm
3. drop  cells in petridish ( I use 3 mL cold HBSS, UVC can not penetrate HBSS if you put too much )
4. I put this petridish on Ice ( I have to run to UVC irradiator room about 5 min)
5. Irradiated with UVC ( MUST open the lid of petridish!!!)
6. quickly split to 3ea T175 cell culture flask (aprox 1million per flask but they will die during I run around with UVC. so I guessed that I have about 50% living cell left) and allow them to grow and subculture to add 6TG at day 3,7,11,16 after UVC exposure....keep them in 37C humidity incubator
7. for the day 3 I expand them ( safe some in 10% DMSO for future use)
8. day 7,day11,day16 I will subculture them and put 100,000 cells per 100mm petridish and immediately add 5ug/mL 6TG with cell medium and allow them about 14 days to form colonies!!
9. after two weeks, I will stain colonies with methylene blue in Methanol (4g/1L) for 10 min and then rince with water.
10. and see if how many colonies you have
GOOD LUCK

------------------
My results
My control ( with out UVC) -- 0 colonies
day 7 after UVC I got 4 colonies/ million cells
day 11 after UVC I got 20 colonise/million cells
day 16 after UVC I got 10 colonies/million cells

This is just my first time results, I will try to repeat them

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Question??

Does anyone know if this mutation (HPRT-) frequence will be decreased after some certain of time?

Such as on day 16 after UVC, the number of colonies are decreased from day 11 like 50% or it was my error technique during counting cell or pipetting that made number of colonies drop like that.

//Tai

before taking images, try to wipe out ECL liquid.

Have you tried to take image one by one per membrane? sometimes, some image which was very light signal could increase background of the membrane which very strong signal. For some reson, the camera try to compensate the signal and try to make all the images to be the most clear.