Hello everyone,
I want to see HMGB1 release to cell culture media to detect the necrosis levels. For this reason, I have used both lysates and cell culture media to make Western Blotting. But my media samples have smeared and have not run properly. You can see in the picture, the last three samples are media.
What do think about this problem? Is it because of the FBS? I thought that maybe I have too much HMGB1 but the first band of these three is the control group. So I do not expect any release in this sample but the view is the same.
Thank you in advance!
Hi Gülşah,
you are right - the smear is due to the serum albumin in the cell culture medium. You could try to use serum-free medium (wash the cells with PBS to remove any traces of serum, then culture the cells in, e.g. OPTI-MEM over night or up to 24 hours). You can use this conditioned medium directly for western blotting to detect your proteins
Good luck with your experiments,
Christian
Hi Gülşah,
you are right - the smear is due to the serum albumin in the cell culture medium. You could try to use serum-free medium (wash the cells with PBS to remove any traces of serum, then culture the cells in, e.g. OPTI-MEM over night or up to 24 hours). You can use this conditioned medium directly for western blotting to detect your proteins
Good luck with your experiments,
Christian
It is nicely explained by Christian Wechselberger . I just want to add that run the gel completely and also probe it with ACTB or GAPDH.
Dear Gulsah,
In addition to nice suggestions above, I want to tell that since secreted protein is very low you might end up with no bands if you run the media directly. you need to concentrate the media to atleast 10-20X and then run it or isolate protein by using methanol/chloroform method. Then only you can get a decent profile of protein bands from secreted media.
Hope it helps,
Ajay
Dear Christian Wechselberger probably you are right about albumin -and also other serum proteins-. I have forgotten to say, this is a gradient gel between %4-12 and I think serum proteins have deadlocked because of their relatively high kDas. Unfortunately, these samples were from an old experiment so it is impossible for me to make them serum-free. I want to ask one more thing; Do you think that if it is work when I use a normal gel with a percentage like %7,5-10?
Thank you!
Dear Pritam Sadhukhan thank you for your answer. I could not run the gel completely because there has been something on my other gel during the running and the dye had started to getting spread at a point... So I have obligated to stop the running otherwise my samples also would be face with the same problem. And after stripping process, I tried beta-actin control.
Thanks again, have a nice day!
Dear Ajay Kumar I have already concentrated the media samples with special columns. But I do not know what you intended by methanol/chloroform method. Could you please explain more? I made the standard Bradford method and load all of them directly. I wonder if have I missed something?
Thank you for help!
Hi Gülşah,
concerning albumin proteins - their MW is around 70kDa, you will always have an overloading-issue if you load complete serum on a gel no matter what gels you use (either gradient or e.g. 7.5-10%).
If your protein of interest is larger or smaller then 70kDa then you could try a MW-cut off column (size exclusion) to enrich for your protein.
If you have more than one antibody to your protein maybe also immunoprecipitation or an ELISA approach could work?
Best regards,
Christian
Christian Wechselberger thank you for your answer. It helped me a lot. I will consider your remarks.
Kind regards
Dear Gülşah Torkay,
Generally the secreted protein is not in pure form and contains lots of salts as it is in media and you concentrated that which eventually concentrate salts also in your sample. Best method to obtain pure proteins is precipitating proteins from concentrated media and methanol/chloroform method is best one for that as it gives you a high purity protein sample devoid of any detergent contamination like SDS or triton which are generally used in TCA precipitation. So best way is to isolate your protein using this method from concentrated media.
Bradford method is not a preferred method for protein measurement from secreted media due to sensitivity issues. Best working method is BCA and it gives a right estimate of protein concentration.
Hope it helps,
Ajay
Ajay Kumar thank you very much for your answer. I will consider your remarks.
Kind regards
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