I'm using the 15% hand casting gel for a 9 KDa protein, later a 4%-20% hand casted gradient gel. This problem the sample band became a line, and cannot be recognized which is which. It happened on every sample including ladder.
So I re-prepared all the casting buffer I used and checked with the Tris-HCl pH. Everything seems correct. After several times of SDS-PAGE, I found 10% gel, 12% gel can run nicely. But when percentage goes higher, the spread problem appear. It's hardly to believe that the casting buffer is not proper.
The running is voltage constant at 80V. The current is 25mA for one gel and dropped to 10 mA in the end. It took 2 hour for 15% gel. I tried 80V for stacking gel, and increased to 110 V for resolver gel or 80V all the way. The spread problem is there.
I really want to know whan happened on my 15% SDS PAGE gel. REALLY! Please give me some suggestion, ideas or anything concern that come to your mind. Thank you so much!!
This is my 15% gel and 5% stacking gel recipe.
15% gel: Distilled water 2.3ml, 30% Acrylamide 5ml, 1.5M Tris pH8.8 2.5ml, 10%SDS 100ul, 10% APS 100ul, TEMED 4ul.5% stacking:Distilled water 2.7ml, 30% Acrylamide 670ul, 0.5M Tris pH6.8 500ul, 10%SDS 40ul, 10% APS 40ul, TEMED 4ul.
The APS is freshly made before casting gel. 30% Acrylamide is mdae with 29.2g Acrylamide and 800mg N’N’-bis-methylene-acrylamide.
(I showed two pictures below, one is the western blot for beta actin. There supposed five samples, but they form a horizontal line. The next one is 4%-20% gradient gel. I randomly chose a 20ug protein sample. Loading volume is 20ul. It's clear that the lane became wider and wider as it ran towards to the bottom.)
This is very simple. Probably you did not loaded all the wells. If you do not have enough samples just load the equal amount of the loading buffer in all the wells.
Hope this solves your problem.
I tend to use a 4% Stacking gel: Distilled water 2.975ml, 30% Acrylamide 670ul, 0.5M Tris pH6.8 1.25ml, 10%SDS 50ul, 10% APS 50ul, TEMED 5ul.
and the 15% Separation gel: Distilled water 2.2ml, 30% Acrylamide 5ml, 1.5M Tris pH8.8 2.6ml, 10%SDS 100ul, 10% APS 100ul, TEMED 10ul.
Running buffer: 150mM Tris, 1.92M Glycine, 1% SDS, final pH 8.3.
I run at 180 or 200V. 80V seems a bit too slow and could be the reason why you have diffusion. Try to use smaller wells as well. An other thing to try is to dilute your sample, if you want to do a western blot you don't need lots of protein on the gel.
I would think that a 9 kDa protein is too small for a 15% gel. We have used tricine gels with better results for such small proteins.
The Coomassie stained gel looks fine to me, the resolution is very good.
Hi there,
The lateral diffusion you are talking about is not a problem as your Coomassie staining looks good (samples remain separated from each other and resolution is OK). If you want to limit this lateral diffusion, you might load less protein and load sample buffer only in empty lanes but the result will be close to what you have already. I think your problem is the WB. Does the membrane correspond to the gel? If yes, you shouldn't get the band everywhere as you haven't loaded all the lanes... Does this band actually correspond to the MW of actin? I'm very puzzled about this result and I would suggest you to stain the membrane with Ponceau Red right after the transfer to check the actual protein profile present on the membrane.
Hi,
I agree, your SDS-PAGE looks fine! Bands detected by WB should have corresponding position on the membrane, they can't spread out to a line during transfer. I think the line on the WB membrane more is related to an impurity in the gel as it covers the lane loaded with markers. Beta-actin is usually sitting in the middle of the gel/membrane. Have you probed for your 9 kDa protein or how do you know it is not there? What pore size of blotting membrane are you using? Such a small protein can easily run through during transfer, I would recommend 0.1 or 0.2 µm.
I appriciated all of your help. I got a lot of good suggests, and feel like have some direction to work with.
I will lower the loading amount, at least for beta-actin. For my protein of interest, I'm afraid lower the loading amount will affect the final western blot signal. I try to limit below 40ug. and yes I load the 1* loading buffer into the emtry lanes.
Right now the 9 KDa protein didn't overexpress in our cell lines. So the its western blot only show the one positive control. I'm culturing the new transfected cells.
The membrane I used it's 0.2 um PVDF. Transfer condition is 75 V 60 min. In case the small protein ran throught the membrane.
Again thank you so much!!
There may be trace contamination of your sample buffer by your protein sample. Notice that every lane is not only Western positive but also the band intensity varies slightly between lanes at what I assume is 9 kD (need mw markers), You also loaded this sample buffer in all lanes, right? A trace contamination probably wouldn't show up on the Coomassie stained gel but certainly on a WB. I've seen this in the past when the same pipet tip was used for every sample or when the blue sample buffer became contaminated, Also notice that there is some extremely high mw material that didn't migrate out of the bottom of the sample wells. Are you running your samples non-reduced vs reduced? By the way, did you run a WB with secondary antibody only, no primary, as a negative control for cross-reacting host proteins?
something wrong with your techniqe try it again several times
Have you sorted the problem? Have you tried with new Temed? Higher percentage gel probably wasnt catalysted well with old Temed.
The problem still there. I decrease the loading volume to 15ul. Also increase the running voltage to 140, and only run 1h. The smear appeared the last 20 mins.
The APS and Temed are fresh, they work very well when I use biorad fast cast kit.
Hi,
I am also facing a similar problem. If the problem is not solved yet with you, the reason for lateral spread of protein leading to fusing of bands could be that the Ionic strength of sample lower than that of gel (if other obvious possibilities are obviated) (This is something I gathered from this site: http://www.bio-rad.com/en-us/applications-technologies/performing-protein-electrophoresis) .
Let me know if this works.
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