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Are certain tissues prone to uneven loading? Issues with intestine. - (Feb/25/2014 )

We are primarily a skeletal muscle lab but have collected other tissues (liver, intestine, adipose)...not exactly on a 'whim' but without much research into whether they should be handled different from muscle. I have successfully run liver and skeletal muscle western blots with even loading.

 

We collect all tissues in cell lysis buffer with HALT protease inhibitor, homogenize with inert beads and spin down @ 10K g's for 20 minutes at 4C before collecting supernatant. We use BCA to measure protein concentration in the supernatant, then dilute with water to reach the same concentration for each sample before mixing 1:1 with laemmli. We then boil for 10 minutes. Before loading into our pre-cast Tris HCL gels, we vortex every sample (and we do this for the BCA and dilutions...lots of vortexting). I load the same volume into each well, which *should* result in the same ng of protein in each well. Gels are run at 150V for 90-120 minutes and transferred cold at 100V for 1 hr. 

 

I have small and large intestine which was handled the same way, but for the life of me I canNOT get even loading. I have redone everything from the BCA onward 3 times. (It may even look worse now). I have sample that has not been diluted, but I no longer have actual intestinal tissue. 

 

We use ponceau staining to determine even loading. Skeletal muscle and liver are clearly even across the gel, and intestine is just all over the place. I would say the middle samples tend to end up with the biggest blots after I ponceau stain.

 

I can certainly invest in a loading control and compare densiometries to come up with a value, but I can't publish these ugly blots! :(

-phrustrated-

Intestines often have things that will digest the protein really really quickly - they need lots and lots of protease inhibitors added - I'd go with about 3x what you would use for other tissues.  You should also freeze any lysate at -80 as soon as possible to prevent the degradation - though some will still occur under these conditions!

-bob1-

if what bob1 says doesn't resolve your problem then you may want to reduce boiling time or temperature (do you see a pellet when you spin after boiling?)

-mdfenko-

if what bob1 says doesn't resolve your problem then you may want to reduce boiling time or temperature (do you see a pellet when you spin after boiling?)

 

We actually have never spun after boiling. I will try this since I've seen it in other posts as well. It is currently my only option as my PI wants me to hold of off doing anything to the unprepared lysate yet.

-phrustrated-

Intestines often have things that will digest the protein really really quickly - they need lots and lots of protease inhibitors added - I'd go with about 3x what you would use for other tissues.  You should also freeze any lysate at -80 as soon as possible to prevent the degradation - though some will still occur under these conditions!

 

They have been at -80 the entire time. If I have lysates in lysis buffer currently, is it too late to add more protease inhibitor? 

-phrustrated-

Probably too late - the degradation happens pretty fast.  IIRC, gut lysates stored for 24 hours at -80 in normal lysis buffer + inhibitors will show quite a lot of degradation.

-bob1-

Probably too late - the degradation happens pretty fast.  IIRC, gut lysates stored for 24 hours at -80 in normal lysis buffer + inhibitors will show quite a lot of degradation.

Ohhh, bummer! They had been in -80 for close to a year by the time I got to them. Thank you for the help. I guess I'll try the spinning-down thing, and aside from that I guess all I can do is compare the expression to a loading control. Guess there will be no pretty pictures of blots in my manuscript. tongue.png

 

Come to think of it, this might be why they look even worse than when I started...

-phrustrated-