Though a separate band pattern is visible, there is a huge smear in the lane. The protein is not overloaded. My sample is rich in polyphenols and polysachharides. I have done TCA precipitation and the pellet was not completely dissolved. I used centrifuged supernatant after dissolving the pellet in order to remove the aggregates.
What could be the problem? How do I clean the smear?
Should use fresh tank buffer at each time, The SDS in the sample loading dye may be get precipitate during storage time. If its possible use little amount of DTT in your loading dye. Increase the volume of glycerol also will leads to sharp bands. Try the best. All the best
I agree with Krzysztof. I had also face similar problem. I used acetone and its worked well. you can try this .............Use Chilled acetone 1:3 (kept in -80 degree C freeze for 30 min before use) for precipitation and kept in -20 degree C for overnight and centrifuge at 10000g for 30 min. Then dissolved in your prepared buffer and go for SDS page.
Depending on how the smear looks like (a picture would be helpful) it might also be that your sample is not well denatured. If while loading the sample you can see it is viscous try to increase boiling time.
PEG can also be used for the precipitation of proteins. As suggested by Sikorski, it could be that the sample is not denaturated too. Try denaturating it at 105 degrees for 15 min.
Agreed@Lekha. Boil the sample to denature properly (I mean respective samples in apendrop and then heat by holding in boilig water) prior to loading.
Since you have good separation of proteins but still smear at the back (though a gel image would be helpful to clearly decide the problem), it could be a problem of in-sufficient washing. You should either wash it better and dissolve it completely, or change the method to precipitate. TCA pptn usually gives this problem.
Dear Amit,
as peviously requested by others, a picture would be of great help. Smears, dots etc may also be caused by old contaminated skim milk (replace), blocking agent not properly dissolved, the ECL fluid is getting towards the end (filter solutions), antibody is grainy (spin antibody and remove liquid into a separate container), invisible gel remnants on the membrane (wash membranes well, use in-date gels, if home made gels is used, use fresh APS).
Thanks for the suggestion, I could upload image but I dont have right now with me. I will try the other precipitation method as well.
hi,
I think your problem is the concentration of gel loading die (Bromophenol Blue). you can use the die with equal volume (1:1 ratio) of your protein sample and it may clear your problem. At the same time you extract the protein with Phosphate buffer saline it will make the good protein separation for SDS-PAGE.
it seems the protein content is bit high that you've loaded to the gel. so try with loading the little diluted sample. if it still comes out with the same results then your protein might get denatured. adding DTT in 'Sample Buffer' also a good way to avoid of getting smear on gel.
Have you tried polyvinylpolypyrrolidone? It is a solid and can adsorb the poly phenols. The binding of the polyphenold to your proteins could contribute to heterogeneity.
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