I’m trying to analyze insoluble proteins from mouse brain. Basically, I extract with RIPA, then take the pellet that’s left over and extract with a urea buffer (7M urea, 2M thiourea, 4% CHAPS, 30 mM Tris, pH 8.5).
When I do a Western blot, the RIPA fractions run perfectly, but some of the wells with urea fractions streak out on the blot (lane 6, ponceau #1 attached, all lanes contain urea fractions from different mice). The streaking occurs from when the proteins first start to enter the gel. The most of the loading buffer is pulled into the gel, and some of the protein stays in the well in trickles out in a line from the center of the well.
One time, I loaded each urea fraction in 2 different gels at the same time. A fraction would run fine in one gel, and then the exact some fraction from the same tube would streak in the other gel in the same rig.
I’m using 4-12% Bis Tris NuPAGE 15 well gel, 1X MOPS SDS NuPAGE running buffer, and NuPAGE LDS sample buffer. I run at 100V for 30 min then 150V for 45 min.
After playing around with different ratios of protein in urea, distilled water, and 4x LDS sample buffer, it seems 5 uL of each of the aforementioned seems to work best. However, I’m still seeing some streaking, and the even lanes that aren’t streaking don’t seem to be running perfectly (ponceau #1).
Other things I’ve learned:
If I bring the LDS sample buffer to 2x final concentration, the loading buffer looks like it’s coming out of the sides of the wells as it enters the gel and there is strong banding on the sides of each lane (ponceau #2 attached).
Bolt gels are absolutely terrible. Every lane streaks out when I run the samples on a bolt gel.
Any advice or guesses as to what may be happening would be greatly appreciated! Thanks!
I'm not heat denaturing the proteins because they're in urea. Heating in urea causes carbamylation and degradation of proteins, and it degrades the urea.
The detergent used in the urea buffer (CHAPS) is not the good one for a western blotting. CHAPS works very well but CHAPS is not charged and can not be use for western blotting. You need to replace CHAPS by a charged detergent such as SDS. You could try to increase the concentration of the sample buffer to replace more CHAPS by SDS. In fact, I will prepare my own sample buffer and not the one of LifeTech, with more SDS (something like 10%).
Do you add DTT (and not b-mercapto) in urea lysis buffer. It might be necessary to solubilize more the proteins.
Do not heat proteins in urea, for 2 reasons : urea do the same job than heat and because urea is not stable and might chemicaly modify proteins.
Thanks for your response Stephane.
I'm hesitant to replace the CHAPS because the urea buffer I mentioned is very widely used in my field. It's a good idea to try to prepare my own sample buffer though. The NuPAGE one uses LDS instead of SDS and has a very high glycerol concentration so that might be causing some of my problems.
I haven't tried adding DTT, that's a good idea. However, the proteins seem very well solubilized in the urea buffer. It's just when I put them in the western blot well that I seem to have problems.
I'm not heating these fractions. I just mix them with the LDS sample buffer and let them sit for ~5 min at room temperature before loading.
Thanks so much for your advice! I'll try a new sample buffer and DTT.
What temperature denature the proteins ?, since it is known that when you have high levels of these proteins tend to warm them add
I'm not heat denaturing the proteins because they're in urea. Heating in urea causes carbamylation and degradation of proteins, and it degrades the urea.
Lindsay, had a ? fo ryou. How do you antigen retrieve your 40um sections?
Hi Partha,
I use the protocol below as a step before I start my regular IHC or IF protocols. Using 0.3% Triton-X your blocking buffer is an important step. The sections look like they have rough surfaces if you don't.
http://www.ihcworld.com/_protocols/epitope_retrieval/free_floating_sections.htm
I only use antigen retrieval if an antibody staining is not working well. For most antibodies, I've found no improvement in staining with antigen retrieval, but there are some that need it to work. I think antigen retrieval is more important with paraffin imbedded tissue. I've heard from multiple sources that frozen tissue tends to retain epitopes better. Hope this helps!
-Lindsay
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