SDS-PAGE running buffer exhaustion

iam doing SDS PAGE for two times, in both i didnt get any sort of bands in the gel, even the gel is not taking the staining dye properly, it is not evenly distrubuted it leves some stains here & there.. iam using 30% acrylamide mix that prepared 4 months back..how can i resolve god gel.. can any one help me..
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i have been running SDS PAGE and i have used running buffer 3 times wihtout any problem. My problem comes during wet transfer ( i use fresh transfer buffer verttime) , but sometimes there is smudging on the PVDF membrane, i checked the gel with commassie and the separation was good.can anyone help me out, has anyone faced a similar prolem with wet tranferonto PVDF
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Your problem lies probably with glycine. It may be degraded. please try a fresh glycine. Also check the SDS.

Please tell me: I am working with SDS PAGE, I am getting a problem of streaking in the bands. what might be the problem? I am using protease inhibitors PMSF and EDTA.
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I am thinking that you have used Tris.HCl, ie, tris hydrochloride (or some salt form of tris) instead of the basic form Tris. Tris.HCl would give typically pH 7.5. Please check.
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I am working with non naturing PAGe. Unfortunately for the past thre times, the pH of the running buffer after preparation is only 7.5.I have taken 3.0 g Tris and 14.4 g glycine in 1 L milliQ water. Does any body can suggest what is the reason? all the chemicals are elctrophoresis grade.
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quote By k.sudheshna on 01-May-2007
iam doing sds page for the plant proteins the bands run linearly upto the half of the resolving gel
there is a change in colouration(turn yellow)and run unevenly. what could be the reason. suggest a solution
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I was having same problem.I prepared fresh gel loading dye and tank buffer.Dont adjust ph of tank buffer with hcl.
BTW does your gel take longer time to run...
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I HAVE BEEN NOTICING VERTICAL AND HORIZONTAL STREAKING AFTER DESTAINING THE SDS GEL. EVEN WITH A SPACE BETWEEN LANES I HAVE CONTINIOUS RUN ON LINES. I HAVE CHECKED PH OF SOLUTIONS AND BUFFER WHAT MAY BE THE PROBLEM?
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my marker is getting resolved but my protein is not resolving at all. what could be the possible problem?
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iam doing sds page for the plant proteins the bands run linearly upto the half of the resolving gel
there is a change in colouration(turn yellow)and run unevenly. what could be the reason. suggest a solution
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Hola...

Soy estudiante y estoy trabajando en western blotting... requieron una pàgina en la cual revele informaciòn descriptiva de las reacciones bioquimicas e inmunoquimicas desarrolladas en la tecnica.

muchas gracias.
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Hi,
I have the same problem with Prasanna:Lately, when I do Western blot, the marker bands and the protein bands are not resolving properly. The smaller bands appear to be fused with one another, while the bigger bands are better resolved. Untill now, all my Western blots have worked properly. Could anyone please suggest me where I was wrong?



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Hi all,
I'm prasanna. Presently i have been working to standardise the electrophoresis and western blot. My problem is, the marker bands are not resolving properly. they appear to be fused, In the similar way with the protein bands also. I'm using all new buffers. Can anyone suggest me where i was wrong. Its urgent.
With regards,
prasanna.
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my protein (14kd) never gets in the sds gel..it always remains near the stacking region..can anyone plz let me know whats the problem???
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i m doin sds page for long time, im not gettin good or fine bands. so how do i get it.
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Can i reuse SDS gel again
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First and foremost, all buffers should be made up of electrophoresis grade reagents(biorad or FisherSci) in highly pure water (MilliQ water). If gels do not stack properly, it is best to prepare fresh stacking buffer (usually the pH fluctuates over time). If the pH is not at 6.8, then the proteins fail to stack, and you get poor resolution. If running the gel with constant current at a high percentage gel, you get what seems to be a blob like band while running, although the gels do separate efficiently, the voltage has reached for me at about 300,which is not good for the system. Always remember, at constant current the voltage increases over time, but it is best to run at constant voltage for high percentage gels (15 and 20%); the current decreases over time. As already known, it is best to use Tris buffer and adjust the pH with HCl. I have placed my electrophoresis buffers (acrylamide, resolving and stacking buffers) in the refrigerator, and I've never had any problems.

For the protein band that disappears during coomassie destaining, try silver stain to see if it really appears. For coomassie, try fixing proteins for a longer period instead of 1hr, especially if the protein concentration is too low. During the destaining process, if gel was not fixed, then there is loss of protein, meaning diffusing out of the gel. Basic protocol, fix, stain and then destain.

For the ghost bands at 60-62kDa, usually that is characteristic at times of BSA present in protein samples. For example, when purifying protein, when the time comes for storing purposes, then BSA, DTT (1mM or less), or even 20-50% glycerol are added to the sample as protein stabilizers.

Protein at 14.5 and 18.6kDa. Usually, if its a commercial antibody, they sometimes let you know if the ab can detect two bands (elongation factor from Upstate detects two bands one at 53 and another around 36, and at times I've gotten both bands or just the 53kDa band). If it is a purified protein, then there was proteolysis to which the ab might recognize both sizes. If preparing samples for SDS-page, it is best to add the sample buffer immediately, then boil, and not freeze samples first, since that can compromise the integrity of the protein. If at times you get that band and others you don't, then it could be on how you had prepared the sample.

Improper polymerization usually suggests that prior to adding TEMED and ammonium persulfate, the buffer was not de-aerated. Oxygen inhibits polymerization. The other possibility, the acrylamide is bad, and so make a fresh batch of acrylamide, or the ammonium persulfate is bad. When you make 10% ammonium persulfate, when you add water, you should hear the solution snap, like rice crispies (as funny as it may sound), but that is an indication to letting someone know if the ammonium persulfate is good or not.

If protein bands are diffused, but not the BT-markers, then it probably has to do with the preparation of the samples. Something has to be wrong with the sample buffer, and if not, then let the gel polymerize longer. Also, it could be the acrylamide ( are you using a 29:1, or 19:1, or 37.5:1) I use the 37.5:1 formulation, so try changing the acrylamide formulation to 30% acrylamide:0.8% Bis-acry. (37.5:1 formulation), might work.

Final suggestion, and I may be repeating, make buffers with highest quality (electrophoresis grade) with MilliQ water, filter all solutions with a 0.22µM filter, and place the buffers in the refrigerator.
Prepare samples with great attention, and on ice. DO NOT place the 10%SDS solution in the refrigerator, the SDS will precipitate out, and thus will have to wait until the solution cools to re-dissolve the SDS.
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I had read a book on acidity & basicity of running buffer. Its explain that once running buffer was used, the buffer at anode will become acidic, while buffer at cathode will become alkaline. Therefore, it explained to us that reuse buffer is not good. But, I wonder how this happened?
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hi

i have done some SDS-PAGE and get the band with lateral diffusion so please suggest me what can i do
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hi

i have done some SDS-PAGE and get the band with lateral diffusion so please suggest me what can i do
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hay,

i have some problem while doing SDS-PAGE the seperating gel becomes milky in colour and not get stainned.

plz tell me the reason of that
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Is there anyone to tell me some website about protein analysis technology? please comment!!


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I performed SDS-PAGE to see subunit of purifed enzyme. The problem is the disappearance of protein band when the gel is destained. Both of size marker and purifed enzyme appear even in the step of coomassie staining strongly. but destaining started, they disappear. please comment!!
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My protein bands appear to be be diffused but the biotinylated marker bands do not. What is the reason? Is there any problem with the gel? I prepared my reagents a month and a half ago.....
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My protein bands appear to be be diffused but the biotinylated marker bands do not. What is the reason? Is there any problem with the gel? I prepared my reagents a month and a half ago.....
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Hello,

i always find two or more ghost bands at the size of 60-62kd in my protein gels. i am not able to understand why? i changed sample loading buffer, filtered it and so on, but still the problem is not resolving, pls comment.
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Improper polymerization,why?
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Hi,

I've a problem with SDS-PAGE. Suddenly i find that the proteins are not getting stacked properly & run in an diffused cup shaped way...can anyone tell me whether its becoz some of the components have gone bad or too much of salt.thanks.
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i am able to see a band of exactly 14.5 Kd in my SDS PAGE. it appears some times and dose not appear some time with silverstaining. it never appears with coomasie. if i do a westernblotting of that i am able to detect it with the same antibodies as that against my protein which is 18.6Kd protein. i am not able to judge whether it is an artifact or is it a real band chopped off from my protein? what could be the possible reason of it being chopped off in such a way if true.and if so the case why am i not seeing it every time i run the same sample.
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