Hello,
So I have been struggling to get a successful western blot using MIN6 derived EVs, and it has been a real struggle.
Everytime I isolate my EVs, and after lysing them, I run the proteins in the gel and see not band at all. I use coomassie blue or the stainfree precast gels to check the run.
The ladder shows up fine though.
After lysing my EVs, I measure the protein amount using microBCA, and when I diluted the sample 1/2 I got a final concentration of around 200ug/ml, and when I diluted it 1/10 I got a final concentration of 400ug/ml. This is already weird but I still loaded my sample assuming I had 200ug/ml to be on the safe side, and I used 4x laemmli in order to avoid unecessary dilutions, and using my calculations I should have loaded around 15ug/ml. But the imaging showed no protein at all, and now I am really puzzled. (the first band is the ladder, and I am supposed to see two bands on the left side of it).
Brifely here are the steps i followed:
-Collect media from min6 cells (150ml)
-Centrifuge 500g/10min, collect supernatant, centrifuge 2000g for 20min, collect supernatant,ultracentrifuge 120000g for 90min(4C), keep the pellet and wash with pbs 150000g for 70min(4C).
-Finally I diluted the pellet in 500ul of PBS and store at -80C.
Lysis:
-Take 100ul of my ev sample, put it in a 10k column, centrifuge at 1400g/15min at 4C, add 500ul of 1XRIPA to my concentrate, spin 14000g/15min 4c. Put the column upside down in the tube and spin 2000g/2min. Add 70ul of RIPA and incubated on ice for 15min, then spin again 14000g/15min 4c and collect supernatant, and put on ice until further use.
Gel:
I use stain free anyKd precast gels with 50ul wells, and I use for the prep solution :4x leammli(900ul)+b-mercaptoethanol(100ul), because I am looking for tsg101 antibody. I then mix 1/8th of prep solution with 7/8th of my lysed sample. Heat up at 70C 10min, and load the sample in the well, run at 120v/1h.
I don't know what went wrong.
I trying once running the gel with unlysed EVs, and I got a faint band when I did coomassie blue, so maybe the lysis is wrong, or the initial amount of conditioned media is too low, as I saw some people starting with 1-2liters.
I would be grateful if anyone can help.
Thanks
Dear Sarah Boucenna
did you check the absorbance of your buffers? Are you sure that there is not some reagent in the buffers that affect the BCA quantification?
best
Manuele
Sarah Boucenna at what stage do you do microBCA, I assume after extraction (lysis step in ur Q). Please use diluted RIPA lysate, at least 10 x dilution, and also use RIPA as blank and standard dilutents. Had similar problem with inconsistent protein wuant and in the end when i made all reagents using same diluted RIPA i found i had very very little proteins (possibly some aggregates) and adjusted accordingly by using high dilutions and urea in my loading buffer. if this fails maybe you are losing proteins to RIPA insoluble fraction or you are not incubating long enough. Run an un lysed sample as well, and if all fails use urea. good luck!
Thank you Rabeah Al-Temaimi , i use 1x ripa, but I will try to dilute it 1/10 and see if that makes a difference.
How did you solve the low protein yield? Did you optimize your isolation process? Or increase the volume of conditioned media? Any tip is welcome
Hello Sarah Boucenna I was using 3x 35mm (using primary cells) glass bottom dishes per treatment, I upscaled to 6-8x depending on seeding density and imaging. I resuspended by my pellet in 100-120ul. since your initial volume is higher than mine maybe 250 ul is adequate for you. My ultracentrifugation is a little different than yours too. Basically my isolation protocol was closer to this (https://arep.med.harvard.edu/pdf/Kowal_Church_2017.pdf). But I dont use 10kD columns! It is my understanding these deproteinise samples for small molecule isolation! Hope you find success.
Rabeah Al-Temaimi I was not aware of that issue with the 10Kd, do you mind sharing the sources saying so?
In that case what protocole do you usefor your lysis? Were yu able to get succesful western blots?
Thank you for yoour tips so far!
Sarah Boucenna in our lab we had Abcam 10KD columns (https://www.abcam.com/products/sample-preparation-kits/10kd-spin-column-ab93349.html) it says deproteinizes at 10000g but i dont understand why u use them if you are doing ultracentrifugation, but I realize you flip upside down but with your volume you dont need to concentrate! We only use them for low size molecules and when doing samples for high purity mass spec. My extraction was RIPA buffer and yes we did westerns and they worked well for microsomes and EVs.
All in all you could be misreading protein conc as you thought. Good luck