Hi! Pratima,
The simple reason for the smear in SDS PAGE is because of more sample you are loading. i assume it as your crude sample, for sample preparation pellet 1 ml of culture then dissolve the pellet in 60 ul of Tris (You can use Tris 1M pH-6.8 which you use for SDS PAGE) and vortex for proper resuspension then add 20 ul of 10% SDS and at last Protein loading dye 20 ul. vortex the sample for 2-5 mins then boil nicely for 5-10 mins. Centrifuge the sample at maximum rpm 12000 for 2-3 mins. While loading the sample in gel try to take the upper supernatent fraction from top. start optimization with the less quantity of sample, load 5ul, 8ul then10 ul. Smear you will see only in the condition where the loading sample is more so my suggestion is to load less sample and try above mentioned protocol.
All the best.
You can also give details about your crude sample like which organism you are using for the protein isolation or SDS PAGE?
thanks gaurav for your suggestion
my sample is plant and i m using the sample buffer (120mM tris ph-6.8, 4%SDS, 0.1% bromophenol blue, 20% glycerol) made a head and stored in -20. at the time of use, adding 10% b-merceptoethanol to the sample buffer and then add to the crushed samples--> vortex for 10 min--> centrifuge at 15000rpm for 30 min--> took superntant and heat at 95 for 10 min--> then load 15ul
May be this time i will load the sample at less volume
thanks Kevin for replying,
I am making the gel by myself (13% and 10%) polyacrylamide gel for proteins separation from plant samples
and sorry, i could not understand what do u mean by how fast am i running the gel
using 100v and maximum current (500mA)
I dont think its the amount of protein you load rather how you prep your samples. I routinely load max of 60 ul per well for 1.5 mm gel, and it run and fine and give nice thick bands after western.
you can try adding glycerol to your SDS gel mix?
I ran my at 100-120v for stacking and 160-180v for seperating.
I agree with Kevin and Gaurav, sample preparation is important factor to get clear and sharp bands. Try to estimate your protein concentarion before loading into gel. Try to load 2mg/mL concentration of your crude protein if you are using Coomassie Brilliant Blue R-250 stain.
Good luck..
thank you all for your help. I got the bands on gel by using less amount of sample to be load.
Thanks again to all of you :)
There are two major causes of smearing in SDS gels: 1. overloading of the protein and 2. poor preparation of the gel for example if polymerization starts before you finish pouring your gel and instead of discarding it and starting afresh, you continue. In this case, there will be no continous flow of the gel and therefore your proteins cannot run systematically as required, ending up in a smear.
If your gel is well prepared, then you need to check the concentration of your protein and reduce the quantity you are loading onto the gel.
Hope this helps you. All the best.
thanks Priscillar for your kind reply. I used less amount of sample for loading and i got the band but still i will consider ur suggestion about gel prepration to get good bands
Thanks again
Pratima, several factors can contribute to this kind of banding pattern.
One thing is do you use any protease inhibitor in your protein extraction cocktail? I think the proteins are getting degraded. If samples were not extracted with proper amount of sample buffer then you might be getting partial recovery which gives smear. Also does you sample look brown or slight brown which a symptom of having phenolics and may be you need to add some PVPP (mg quantity) while grinding tissue. Are you storing your samples in -20 right-after harvesting? How much protein you are getting in tissue? Are you using mini-gel or the big-gel apparatus for SDS-PAGE. Usually for mini gel 25-30 µg samples are good enough with current 30 mAMPs. Also if the running buffer is old then it will give you unusual banding pattern. Mainly you need to be careful right from extraction and quantify then and convert the quantity to µg. Also before loading samples you can prerun the gel for 5-10 min. Hope this will help.
It may be due to how you cut the proteins. It can also be too much proteins added onto the gel which can be problematic.
I would agree with Mishra. You should try adding protease inhibitors to your protein solution and also consider checking the current. Also, be sure of the running buffer. Old buffers usually resist current flow and could result in a smear.
First thing that comes into my mind is, you have forgotten to heat your sample or you dont have b-mercaptoethanol/DTT in your SDS buffer. double check your steps and reagents you are using.
thank you all for your kind reply. I got he bands
I was loading much of sample and when i load 10ul and 5ul of sample i got the bands.
Thanks again to all
I am always getting a smear as a result of my SDS page in the lower part of gel(small protein area).
what is the reason?
I think the proteins are getting degraded too. use protease inhibitor cocktail +Sodium orthovanadate + sodium fluoride as inhibitor.
be luck.
Most of the times, the problem is either bad buffers or protein degradation during purification and storage. I would first make sure that these are not happening.
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