What to load in empty well on SDS gel? - (Nov/30/2006 )
Hi!
Could you help me and answer the question. What I should load in empy well on sds gel when running Western Blot. My gels are 12-wells but I need only 9 samples plus marker. Last time I have loaded only samle buffer in water, and also got the band in those lines. It was the same size as for my protein. That made me thinking, is it something wrong with my sample buffer?, or maybe during the loading some of the samples "moved to empty wells"? Hmm, that makes me confused. And one more, do you load dry gels or first prepared everything for running and load when gel is placed and cassete in filled with running buffer?. Thank you for time and help.
Could you help me and answer the question. What I should load in empy well on sds gel when running Western Blot. My gels are 12-wells but I need only 9 samples plus marker. Last time I have loaded only samle buffer in water, and also got the band in those lines. It was the same size as for my protein. That made me thinking, is it something wrong with my sample buffer?, or maybe during the loading some of the samples "moved to empty wells"? Hmm, that makes me confused. And one more, do you load dry gels or first prepared everything for running and load when gel is placed and cassete in filled with running buffer?. Thank you for time and help.
You should load the gel when everything is ready, never whithout the buffer covering the wells.
You are not supposed to see any band where you didn't load a protein sample. You should load sample buffer in the wells where you don't load protein extract
that's strange. I never load empty wells (unless my samples are rich in salt so I load salts diluted in sample buffer)
do you mean that you have a real band in non loaded wells, or that the band from loaded well is spreading to the non loaded well?
i load the wells when it's full of buffer, so I don't spread the sample when I fill the wells with the buffer.
I use enough glycerol so the sample goes stright to the bottom of the well, and I use special tips, that are thin and long, and allows to load directly at the bottom of the well.
Be careful, if you wait too long over a well , the sample will leak, and fall inside the well.
wel.. for empty well i understan that is... empty...like empty.. empty, but weird that u got a band in your "empty" well. could it be that leaked some of the sample of the next well?
Could you help me and answer the question. What I should load in empy well on sds gel when running Western Blot. My gels are 12-wells but I need only 9 samples plus marker. Last time I have loaded only samle buffer in water, and also got the band in those lines. It was the same size as for my protein. That made me thinking, is it something wrong with my sample buffer?, or maybe during the loading some of the samples "moved to empty wells"? Hmm, that makes me confused. And one more, do you load dry gels or first prepared everything for running and load when gel is placed and cassete in filled with running buffer?. Thank you for time and help.
1. May be your sample buffer is contaminated.
2. you overload your sample, and the sample flood to the next empty well.
3. after loading the sample, you wait too long before running the gel and the samples diffuse to the next well
4. may be you forgot which wells you had loaded.
Hope this may help.
if i have extra wells, i prepare duplicate samples - it's just a matter of pipetting an extra 20ul or whatever. i don't trust protein-free sample buffer to run evenly. and you never know when a bubble or flaw in one lane of the gel will screw up a band, in which case that duplicate comes in handy 
do you mean that you have a real band in non loaded wells, or that the band from loaded well is spreading to the non loaded well?
Yes, I have a real band in non loaded wells, it's the same size and everything. Even more, the non-specific bands are present there?
clean the comb.