For western blot sample preparation of mice brain tissue (control and transgenic groups), I used RIPA with stainless steel beads for homogenization. After centrifugation, transgenic samples had a muddy pellet of tissue with steel beads, but control only had the beads and no visible tissue. I used the supernatant of both groups for BCA assay.
Protein concentrations from control group turned out to be great (at least 10 mg/ml). Transgenic tissue concentrations on the other hand were almost 10 times less. Around 1 mg/ml or under. Obviously, it's because the tissues did not get disrupted as well as the control tissues. I repeated the procedure for transgenic samples, nothing changed.
Any ideas and help would be much appreciated. Thank you!
Edit: I am attaching my sds-page gel photo to give an idea of how different the results come out. First four samples are control, last four are transgenic. Each sample is calculated to be 20ug.
Hi Saglik,
Are there obvious anatomical differences between control and transgenic brains? As example, and Hipothetically talking, if the brain of your transgenic mice is 60 % white matter (myelinated axons) while in controls this is 10%, then you'll be seeing more pellet (insoluble fraction) and recovering less amount of soluble protein in your transgenic lysates than in control ones?
Have you evidence of such anatomical difference between your groups?
Cheers,
Hi Sebastián Dupraz
There is no difference anatomically. Also, these mice are 3 months old, they are at the early stage of the disease (Parkinsons), so we do not expect to have such obvious difference. I have tried to homogenize different parts of the brain from both groups, but transgenic tissues always give this muddy pellet.
I am attaching my sds-page gel photo to give an idea of how different the results come out. First four samples are control, last four are transgenic. Each sample is supposed to be 20ug.
Thank you!
Hey, this is indeed weird.
The problem is very likely the extraction but it is not clear why the difference between your controls and transgenics groups given that there's no anatomically difference between them. On the other hand, when you said no differences at all I'm assuming you also checked brain sections as well as stained for aggregates and fibrils, etc. Am I right? Nevertheless, you could try the following: Perform you homogenization as usual but then add regular laemmli buffer (containing SDS/b-mercaptoethanol/DTT, etc) to the tube to reach 1X of it and boil it for 10-15 minutes and check whether you can clear the pellet. You can do the same for the control for comparison. If this works, you'll need to think in harsher lysis buffers (i.e containing sarkosyl, or high SDS or another ionic detergents).
Best,
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