Hi,

You could perhaps try out the following points to help you with better bands:

I hope this helps.

My guess is sample preparation or gel overloading. You do not specify the tissue or lysis buffer. Is the sample fatty/sugary? Is the prep very concentrated? Is there enough SDS in the buffer?

Also, you can try slowing down the electrophoresis (drop the amperage), it sometimes suffices to straighten ugly bands!

Try loading tip for loading samples with concentrated loading dye such as 5x or 6x (Sample volume should be low but concentrated enough). Keep the tip at the bottom end of wells while loading samples. I guess you use regular tips for loading samples.

Is the gel premade one, or you make it by yourself?

Try lowering salt concentration of lysis buffer.  Also decrease volume as suggested by Goodwin. Are you using a stacking gel? Also slowing the gel may help.

Hi abishek,

you can cross check some of the points 1) The sample (protein) volume of desired concentration is between 5-10 micro liters. 2) make samples with 4X dye and adjust final vol to 20 micro liters. 3) use freshly prepared running buffer and allow proper stacking by running gel at slow vol or curr. 4) if your protein of interest is of higher mol wt then run transfer for longer times. 5) the percentage of gel for your protein size needs to be checked as this is very important for good and sharp bands.

Hope this may help you and inshahallah you will get your results soon.

Hi,

You could perhaps try out the following points to help you with better bands:

I hope this helps.

I would not worry too much about volume if stacking gel is good. check the pH of both stacking and resolving mix as it is the sharp change that causes the stacking as well as the difference in acrylamide conc. ! BTW, I successfully ran 50 ul samples on freshly cast gels.

1. First, what running system are you using?  Are you using the mini-protean or criterion system from biorad?  If you are using the mini-protean system, you need to make sure that the running chamber has enough running buffer and is not leaking. 

2. Are you making your own gels or buying them?  I use precast gels from biorad and they work great.  They cost more than making your gels but make up for it in convenience and flexibility.

3. Are you making your own running buffer?  Like above posts have stated, the pH and SDS concentrations are very critical for proper running conditions. I use the pre-made 10x Tris-HCl buffer from Biorad running at 1X. This insures that the contents of your running buffer are optimal without having to worry.

4. Are you loading equal amounts of sample into each well?  I sometimes have problems like yours if there are not equal amounts of sample loaded.

5. What running conditions are you using?  ie, what is your voltage.  You might want to check to see if the wiring on the running unit is working properly.  On an old unit of mine, the wiring was coming loose on one end and caused irregular patterns. 

Good luck!

There are about 10000 reasons for what you got, in order to answer your question you should first answer the following by yourself: 

1. Did you have enough Running buffer?

you results suggest that the current didn't go through the gel evenly, that why you get the M-like shape....

if that's the issue....your problem solved ... just re run the samples  with more running buffer and check it from time to time to make sure it didn't leak...in order to avoid it, I usually fill the tank all the way above the gel... 

2. Is it a freshly homemade gel or precast one...? 

if it is homemade , then you probably should clean your casting system better, first with DDW and then with 70% Et-OH prior to pouring the gel, dust grains might effect the gels polymerization and might cause such result.

if your gel is a one you buy from bio-rad, make sure you remove the sticker at the bottom of the gel.... (it happens to everyone at least once... ;) and can cause such results... from experience ... 

3. From the signal strength it seams there is no problem with the loading and the level of protein ... don't waste your time on this factor, the transfer look good, there for next time run your gel in lower voltage... that will give you good strait line (70-100v for 1-2hrs- bio-rad recommend 300v for 30min..) that is in my experience after doing over 3000 gels  

hope that help you ... 

and always 

keep an eye on the gel... that way you can fixed things on the go if something doesn't look good

Good Luck

Hi Abhishek, this could be a problem with high salt in your sample, as many have suggested.

A quick way to find out is to run the MW standards in your sample buffer. If there is no difference between the MW standards run in regular SDS-PAGE sample buffer and MW standards run in your sample buffer, then something is going on with your sample.  

I guess the pH or solvent of protein is the effective factor. Please check the solvent in which the protein is resolved. It is better the solvent be non-polar (the best is water or PBS).

PH of Solvent is not Optimum Plz chek solvent .

plz check the pH of running buffer and gels. Also, the strength of running buffer. I recommend BioRad or GE instruments.

I think you are running gel with used buffer repeatedly. pl use fresh buffer. 

Possible reasons/solutions

a) Poor polymerization around sample wells. Increase ammonium persulfate and TEMED concentrations by 25%.

b) High salt concentration in sample. Remove by dialysis, Sephadex G-25 or any other desalting column or by Amicon concentrators.

c) Excessive pressure applied to the gel plates when the gel is placed into the clamp assembly. Do not overtighten the screws on the clamp assembly.

d) Uneven gel interface. Use a spirit level to make sure the gel apparatus is even. Overlay separating gel with water carefully.

e) Uneven heating of the gel. Either use a cooled apparatus or reduce the current at which electrophoresis is performed.

f) Insoluble material in the gel or inconsistent pore size throughout gel. Filter gel reagents before use and ensure that the gel mixture is well mixed and degassed before pouring the gel.

g) Insoluble material in the sample - centrifuge the sample and apply only soluble material to gel in wells

Mostly running buffer is old or repeatedly used. Check pH . what mV did you run the gel?

Do a acetone extraction of your protein prep. Get rid of the solvent. Air Dry the protein pellet. Dissolve the protein in SDS-Loading buffer and then try running the gel.

Agree with running buffer problem when repeated used or faulty diluted, but please also check SDS-loading buffer make sure at 1 x  final working concentration.

Looks like a high salt in the sample to me. Considering your markers have moved well, I dont think it is a gel or running buffer issue. Try desalting your sample properly post  processing, and ensure that it is optically clear prior to loading. Buffers must be fresh any way.

an excess of detergents like Triton X-100 is a frequent cause of bands like those, especially for low molecular weight proteins. If that's the case, buffer exchange or dialysis, or even precipitation-resuspension of your samples can help

In addition to the suggestions above (which are excellent), I would suggest matching your buffer system to gels and evaluating the voltage or current applied to this gel during separation.  One other thing, temperature may increase during electrophoresis and lead to this sort of pattern. 

Generally these "smiling or crying" effects are observed  in the SDS-PAGE gel due to any of these reasons- Loaded sample( preparation method- excess detergent or salt), Gel running conditions(high voltage etc), or pH of the resolving and stacking gel before casting. So these may be taken care.

This events normally occurs when pH of resolving and stacking gel are not adjusted. Once our SDS used in lab was expired and our SDS band was wavy. Checking your SDS also may help.

Is this extract or total cell lysate? We used to have that sometimes when there was way too much DNA in the total cell lysate sample. We then added Benzonase/Cyanase to the total cell lysate before mixing with SDS-Loading buffer to digest the DNA. Then the samples were running fine again.

Thank you all for your wonderful suggestions.... The blot is now coming fine!

I tried changing the conditions one by one as suggested by many of you, and have realised that the problem was with the pH and some salt precipitation in the buffer used in gel preparation....

By the help of all of you my bands are now behaving nicely :)

Thank you all