Help!- SDS-PAGE- distorted lanes (photo:)) - (Jan/13/2007 )
Hi everybody,
I have a problem with protein resolving on PAGE gels using denatured conditions. I have never done such experiments, and now I can't start with this. My gels (and subsequently, blots) seems as on attached photo. I read carefuly advices on this forum, but unfortunately, I haven't found something passing to my gels. So, please help!!
ps. Note, that stacking gel is removed on the photo and visible "lenses" lies just in the upper part of resolving gel. What happens with lanes 3,5 and 6, while e.g 1 and 2 are acceptable???
I use a 6% and 12,5% acrylamide in stacking and resolving gels, respective. I overlay poured separating gel with butanol (water saturated) or with isopropanol. Both gives the same (ugly) results. I though about salt in samples, but the lanes 1,2,3 contains the same cell extract. Sample in lane 6 was treated with spin column in order to desalt, but it don't gave any better result. I always remove air- bubbles from down part of glass plates. I centrifuge the samples 10 000xg for 5' before loading (to avoid any solid particles).
I suspect an old SDS (fresh made, but from an old powder) or uneven boundry between stacking/resolving gels. Should I wait longer for polymerisation ( I wait usually about 20 minutes, till the gel is visually polymerized)???
Any other ideas?
Thanks in advance for any help 
I have a problem with protein resolving on PAGE gels using denatured conditions. I have never done such experiments, and now I can't start with this. My gels (and subsequently, blots) seems as on attached photo. I read carefuly advices on this forum, but unfortunately, I haven't found something passing to my gels. So, please help!!
ps. Note, that stacking gel is removed on the photo and visible "lenses" lies just in the upper part of resolving gel. What happens with lanes 3,5 and 6, while e.g 1 and 2 are acceptable???
I use a 6% and 12,5% acrylamide in stacking and resolving gels, respective. I overlay poured separating gel with butanol (water saturated) or with isopropanol. Both gives the same (ugly) results. I though about salt in samples, but the lanes 1,2,3 contains the same cell extract. Sample in lane 6 was treated with spin column in order to desalt, but it don't gave any better result. I always remove air- bubbles from down part of glass plates. I centrifuge the samples 10 000xg for 5' before loading (to avoid any solid particles).
I suspect an old SDS (fresh made, but from an old powder) or uneven boundry between stacking/resolving gels. Should I wait longer for polymerisation ( I wait usually about 20 minutes, till the gel is visually polymerized)???
Any other ideas?
Thanks in advance for any help
Please, give me your exact formula for both gels, your sample (SDS) buffer, running buffers and which stayning is it? Estimate the amount of protein in each lane... so all infos, than I´ll try my best...
First, looking on your MK, there are some nice lanes on top, than some not sharpe bands, and the lower ones run to the right. Therefore I think that your gel is not polimerized in one and somethings goig wrong with the running buffer and or the running chamber (which one do u use?)...
Give me some more details, ok?
I have a problem with protein resolving on PAGE gels using denatured conditions. I have never done such experiments, and now I can't start with this. My gels (and subsequently, blots) seems as on attached photo. I read carefuly advices on this forum, but unfortunately, I haven't found something passing to my gels. So, please help!!
ps. Note, that stacking gel is removed on the photo and visible "lenses" lies just in the upper part of resolving gel. What happens with lanes 3,5 and 6, while e.g 1 and 2 are acceptable???
I use a 6% and 12,5% acrylamide in stacking and resolving gels, respective. I overlay poured separating gel with butanol (water saturated) or with isopropanol. Both gives the same (ugly) results. I though about salt in samples, but the lanes 1,2,3 contains the same cell extract. Sample in lane 6 was treated with spin column in order to desalt, but it don't gave any better result. I always remove air- bubbles from down part of glass plates. I centrifuge the samples 10 000xg for 5' before loading (to avoid any solid particles).
I suspect an old SDS (fresh made, but from an old powder) or uneven boundry between stacking/resolving gels. Should I wait longer for polymerisation ( I wait usually about 20 minutes, till the gel is visually polymerized)???
Any other ideas?
Thanks in advance for any help
Hi,
i use PAGE gels with 12% acrylamide in resolving gel, these problems appear before or after the migration and which are the pH ? Secondly, in your protocol of SDS-PAGE preparation which are the pH value, the concentration of Tris-HCl, etc...?
Hi,
There are my SDS-PAGE conditions:
1. Running buffer is: tris-base 15,1g, glycine 94g, 10%SDS 50ml, water to 1l.
2. Lysis buffer: 150mM NaCl, 50mM Tris-HCl pH=7.4, 1mM EDTA, 1mM PMSF, 1%Triton x100, 1% sodium deoxycholate
3. 2x Sample buffer: 50mM tris-HCl, pH=6.8, 1%SDS, 12,5%glycerol, 5% beta-mercaptoethanol (freshly added), some bromophenol blue
4. Separating gel: 12.5% arylamide/bis (19:1), 0,375M Tris pH=8.8, 0,1%SDS, 0,09%APS, 0,08%TEMED
5. Stacking gel: 6% acrylamide/bis (19:1), 0,125M Tris pH=6,8, 0,1%SDS, 0,09%APS, 0,08%TEMED.
6. Sample were prepared from HEK293 and swine PK15 cell lines. Loaded amounts were:
lane1: 10ug PK15,
lane 2: 10ug PK15(2x diluted with water),
lane3: 10ug PK15 (5xdiluted with water),
lane4: an old HeLa extract, in which no protein cocnentration was calculated, I loaded just 10ul
lane5: 10ug HEK293,
lane6: 10ug HEK293(2xdiluted with water),
lane7: 20ug HEK293, centrifuged previously on VivaSpin column in order to remove any salt residues.
lane8: MK from Fermentas (I'm in home now, I don't remember the exact name)
All samples was prepared from centrifuged lysates (11000xg/5min), heated at 99deg/5min and warm loaded onto gel.
I used mini-protean 3 apparatus from BioRad at constant 200V (also trying 100 and 150V with the same effect).
Aha. I used running buffer 2-3 times before I threw it out... 
Thanks in advance for your advices!
Hi grzeniu, on tuesday i will check all your buffers against my protocol, on monday I have a job outside.... hope you could wait or there will be someone else with some hints...
Where are you from?
Wait, I see, first mistake is your running buffer, to much of all (i think it is somme like a 2 to 8 fold buffer...)
Here is a correct on:
10X Running Buffer (Laemmli electrolyte buffer)
Glycine 1.92 M 144g
Tris base 0.25 M 36.3g
SDS 1% 10g
all up to 1L
Dilute 10-fold before use. Replace if the final pH is not within 0.1 pH units of pH 8.3.
Where are you from?
Wait, I see, first mistake is your running buffer, to much of all (i think it is somme like a 2 to 8 fold buffer...)
Here is a correct on:
10X Running Buffer (Laemmli electrolyte buffer)
Glycine 1.92 M 144g
Tris base 0.25 M 36.3g
SDS 1% 10g
all up to 1L
Dilute 10-fold before use. Replace if the final pH is not within 0.1 pH units of pH 8.3.
Dear ms-olli,
I will patiently wait for your answer. Fortunately, I also have some lessons tomorrow...
And, ups... of course, the data, which I gave is for my 5x buffer. I compare it with yours one.
Thanks!
Your question- Ich lebe ganz in der Naehe, hinten Eurer ostlicher Grenze... Tschuess
i would suggest you to start your run at 80-100 volts till the proteins are in the resolving gel. They shouldn't enter and can't concetrate well at this voltage. (150 -200)
Second one, seems there are some resiues in your well... Did you eliminate that possibility?
Try 4%stacking gel.
Finally, APS and TEMED conc seem ok for me.
Second one, seems there are some resiues in your well... Did you eliminate that possibility?
Try 4%stacking gel.
Finally, APS and TEMED conc seem ok for me.
Hi,
My topic seems to be resolved!
According to yours suggestion and to other informations founded on web, I tried to change SDS solution (I made it always fresh, but this time I used an other lot) and I also changed my sample buffer (now with DTT instead of mercaptoethanol) and... it did work!!!
I am sending you the photos of my gels tomorrow to enjoy, what a beautiful they are now
Best greetings and thanks for your advices!
As I said,
There is picture of my new gel. Well, it is perhaps not ideally, but much better than the previous one.