How would the fat contamination in the xenopus embryo lysates affect the outcome of the western blot? How could I minimize the contamination? I use the lysis buffer and pipette out the lysate (floating is the fatty layer) after spinning @ 13000rpm but could not avoid sucking a little bit of fatty layer. Any suggestions?
Thank you for the suggestion Mr. Jesse Grey, could you also help me know the problems downstream?
Hello Sanjeev!! This is a common problem I also faced during the lysis of tissue samples from colon of rats. But I found out a very simple way to overcome this trouble and since 5 yrs my Western Blots are more than awesome. So, if you could follow the following steps carefully you would never see any fat contamination in your final lysates. Always make 10% (w/v) lysates of samples.
1. After adding chilled lysis buffer to the samples process them the way you do but keep in ice all the time. 2. After your processing do a harsh vortexing for 35-60 sec for each sample. 3. Centrifuge the samples are 12000 rpm at 4'C for 20 mins. 4. After centrifugation carefully tilt (45') the tubes towards the opposite side of the pellet formed and insert pipette tip (200ul only) inside the clear portion of the lysate and go towards the pellet without shaking the tube. Remember to compress the pipette outside the tube so that you don't disturb the system inside by bubbling. 5. Take out the clear lysate up to ann extent that you can see the pellet is not disturbed and don't try aspirate the whole lysate. 6. Keep the clear lysates and the left out lysates at -20'C for 24 hr-48hr. 7. Take out the lysates and thaw on ice don't check them repeatedly.
8. In the left out lysates with pellets you may add nuclear protein extraction buffer (50-100ul only) and do vortexing for 2 min each 9. Centrifuge all of them again at 12000 rpm at 2'C for 15 mins. 10. Very carefully remove the tubes from centrifuge and place directly in a pre-chilled (4'C) stand. 11. Now, you would be able to see a thick white layer on the top of the clear lysate in each tube (our enemy). 12. Repeat the same step 4. don't forget to compress the pipette outside and use only 200ul tip and suck gently till you see the white layer is coming with the lysate. For nuclear extracts use 20ul pipette tip only. In the last step 12 always hold the tube from the top so that the temperature of the lysate should not rise with your touch and quickly perform it. If possible do in cold room because lipids are very temperature sensitive and the solidified once starts melting quickly.
After taking out the clear lysates aliquot them and keep some at -20'C and some at -80'C (for longer storage). I believe that with this protocol you will enjoy Western blotting.
Use a sample buffer that contains DTT but not B-ME. If you run 2 gels together in a unit run them at 20mAmp continuous and for 4 gels in a unit use 30-40mAmp.
Good Luck!!!!
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