Dear Eden

You have severe vertical smears. You can follow three comments to overcome it:

1. Centrifuge all samples before loading wells. If problem still persists decrease %T of separating gel. These two ways can help to remove sample precipitation. 

2. Dilute sample

3. Reduce voltage by about 25% to minimize streaking.

What is this protein? If this is a cell or tissue lysate you should be expecting this kind of smear. You might get improved resolution using a gradient gel, but if this is a complex protein mixture you should expect to get many superimposed bands.

First, you need to check all parameters of the running a gel (concentration and ratio of Acrylamide/Bisacrylamide, concentration and pH of the stacking gel and resolving gel buffers, concentration of SDS in each of them and in the SDS-running buffer and so on) . If you run a precast commercial gel and use commercial SDS-running buffer you can skip the parameters about the gel itself and the SDS-running buffer. 

Second, you need to make sure that the pH and concentration of SDS in your SDS-Sample Buffer (which you used for your protein sample) are OK. If you are using commercial SDS-Sample Buffer you can skip this step.

Third, you need to make sure that your protein samples do not have high concentrations of salts or detergents.

Forth, if all of the above were checked and OK, try to decrease the total volume of your sample that your load in each pocket. The less the initial volume of your sample the sharper the band of your protein you are going to get on gel.

Hi,

are you looking for a band of a specific size? If you are loading a complex mixture then the different bands may be difficult to resolve. It looks like you have loaded quite a bit of protein on that gel. If you know the size of the band you are looking for, I would suggest seeing if the band at that size is larger or smaller than you would like, and depending on that, adjust the amount of protein loaded.

Also, you might try to limit the time between running the gel and staining/fixing it. My experience is that proteins in a gel may become "blurry" if the gel is left too long in buffer without voltage and without fixation.

Hope this helps!

Also check the conductivity of water you are using for preparation of silver staining solutions; it should be low.

looks like old gel or improper sample preparation...work on that

Hi, try to make dilutions of your fractions or whatever your samples are. Try different percentage of gels (10, 15, 18, 20 %). How is your sample handling? Did you boil the sample before you loaded it onto the pocket? You can run the gel also longer under lower voltage than what you usedhere, then you will  have  a better seperation. I hope this can help

Also silver staining accompanies with background noise. Use a very clean gel holder for silver staining, every single impurity will effect your results. 

HI,

Possible causes:

 Protein sample is only partially denatured

 Protein sample is only partially reduced

 The gel was run for too long

Possible solution:

 Ensure that the protein is fully denatured, use fresh sample buffer, heat longer

  Make sure the proper amount of DTT or  ß-mercaptoethanol is added

  Watch the front dye as an indicator for proper running duration

Hope this helpful

Dear Eden

You have severe vertical smears. You can follow three comments to overcome it:

1. Centrifuge all samples before loading wells. If problem still persists decrease %T of separating gel. These two ways can help to remove sample precipitation. 

2. Dilute sample

3. Reduce voltage by about 25% to minimize streaking.

Thank you all for you comments!  It is most appreciated.

A few clarifications first:

1.  I'm running purification fractions of an in vitro protein expression reaction.  I'm loading 5 ul, boiling them for about 5 min and running gel at about 150V for 1 hour.

2.  This is a homemade gel and homemade buffers, not commercial.  All of the buffers I made recently except for the sample buffer which was made by someone else in my lab probably at least 1 year ago.

3.  This is my first time doing silver staining so I think I may be over developing it.  I am also reusing the same gel holder but cleaning it after every staining.

Sharpness can be dealt by increasing the % of gel (14-16%). Make sure everything is freshly prepared (APS, Running Buffer, etc...).

Also, dissolve appropriate concentration (Try various concentration) of protein sample in sample buffer and make sure you have kept in 95 degree C for 5 minutes after adding the sample buffer.

best of luck!

Dear Eden

How many current (mA) do you use for gel running?

Is your gel run in ice condition?

What is your procedure for silver nitrate staining?