Did you used a freshly prepared running buffer? that can affect protein migration of the proteins. Too concentrated protein may cause problems with protein migration. There is also unlikely possibility that you put the electrode in the opposite direction.

I will follow this topic to get another point of view from other peers..

If you use too much of ammonium persulfate and/or TEMED this can happen. So use good quality reagents and the right concn. to wvoid such problems

Check out all the composition properly..you can follow the sambrok laboratory manual for the further details on sds-page..and if you have used old buffer also it should migrate but in poor form..in my opinion your problem must be in the preparation or may be in the composition of SDS-PAGE gel..try to short it out again.

all the best.

Only reason is no proper polymerization of stacking gel, There may be more artifacts in the stacking gel or Acrylamide may be expired. If you make a solution in larger concentration polymerization is easier, but in low quantity its creates problems. There is another possibility use the sample only after passing through cellulose filters of 44 mu micron size to remove out larger non-protein substances because they obstruct capillary movement of proteins downwards.

Dear Budak

How many times you wait to gel run?

Which V and mA you have used?

Are you see the front dye in gel migration?

Granting that the polarity was set correctly, I would first take a re-look at the buffers, both composition and pH: running, stacking and separating gel buffers, and, very important, at the pH at which your protein was buffered. Since the markers also do not show up, it is clear that there has been no migration. If the electrodes are wrongly connected, this would be easy to see: as soon as you switch the power supply on, you would notice the loading dye moving out of the wells rather than into the gel. Check your buffers, if necessary, make all four afresh and run the markers again along with a known protein like BSA.

You should have to ponder over the basic requirements like maintaining the pH of stacking and separating gel, concentration of proteins, and most important is the connections to voltage source. It must be connected to proper polarity or sometimes there may be problems with the electrodes. Check whether current is flowing through the electrodes or not, there may little bit bubbling near by electrode wires through gel that confirms the flowing of current.

The electrodes were applied to correct position. I saw my protein marker migrated down from the stacking gel after the bottom of the well, but, it got stuck and migrated a bit further after the separating gel. The electrophoresis was set at 200 V, 60 mA within 1 hour-2 hour. The voltage value was set constantly during electrophoresis. When the time's up, nothing color trace of loading buffer was present at the bottom of separating gel. For protein marker, it got stuck at upper phase of separating gel.

I prepared both gel that fix to the size of 10-well gel. The percentage of separating gel and stacking gel is 12% and 4% respectively. My predicted MW of protein sample is around 50kDa. During the preparation of both gels, I double the volume of 10% APS and TEMED from the actual volume since it will not get solidified in recommended time. So, I just double the volume to speed up the time of polymerization and the method works for that purpose. Then, I use non-fresh running buffer to run the electrophoresis.

I would still recommend, make fresh buffers, all of them, and cast fresh gels...could you please tell me the composition, molarity and pH of all the buffers you used?

I agree with suggestions above. Really go back and check everything. Take a close look at your gel as it is running, is the dye front moving into the stacker, more than likely not.

1. check all buffers: check pH, has SDS been added to buffer. Have all the buffers been made up correctly

2. Make sure your percentages of acrylamide-Bis acrylamide in your recipes are correct

3. Sample buffer: is this correct, does it contain the correct components

4. Does you gel apparatus work properly i.e. is there current. Check electrodes

5. is you apparatus set up in the correct manner

6. Never leave a gel to its own devices until you can see that it is running properly i.e. sample runs into stacker: you can test this without sample just load laemmli buffer

good luck

Protein marker is denatured, only pH can hinder the band formation, nothing more

I recently had a co-worker who had the same persistent problem, or at least similar to the one you're describing. When I watched her prepare her gel, it was clear that somehow the stacking gel had physically seperated from the resolving gel. The protein lysates and marker would migrate through the first layer and then smear and difuse at the interface. I wasn't able to reproduce using my own reagents and the problem also resolved for her when using freshly made buffers and carefully cleaned glass plates.

I agree with Ian, If we hadn't watched what was going on once the electrophoresis started the problem would have remained elusive.

An informative resource for electrophoretic anomalies can be found at the SDS-PAGE Hall of Shame ( http://www.ruf.rice.edu/~bioslabs/studies/sds-page/sdsgoofs.html ).

If nothing more its an entertaining read .

use quality acrylamide check the pH of both stacking gel and separating gel.

I agree with Ravi and Ian regarding checking the basics of the SDS setup before getting into conjecturing about which reagent component is the culprit. You will save yourself much heartache by taking this seemingly tedious approach. Strange agglomerations can happen in the polymerization phase of the gel setup; temperature in the lab, a lab mate of mine dropped our box of preprepared gels and all of my bands looked like pqrst waves, even mixing acrylamide sol'n well enough before pouring in between the glass plates is important, maybe skip the "speeding up double volume" step to get the gel to set faster - just make them the day before.

Have you repeated the experiment with fresh buffers whose pH and molarity are correct? Does your power supply really deliver the intended voltage? If your marker proteins do not enter the gel, something is wrong with buffer molarity, pH, gel concentration, or power supply and polarity. To check the power supply and polarity, look for bubbling at lower and upper electrode wires immersed in the buffer. The upper pole (negative) should have twice as many bubbles coming out as the lower pole (positive).

Rise the current to something around 150 mA. By fixing it to 60 mA your power supply will not reach 200 V. I usually set the current to 270 mA to make sure that this is not the limiting factor.

Had this kind of problem once because someone adjusted the voltage to very low settings and my blot (at 220 mA) never reached the current and I blotted nothing ^^

With above suggestions, make sure your buffer between two plates is not leaking, so that your connection stops in the mid way.

The question is why did they not polymerize properly to start with.

Make ammonium persulfate solution fresh every day. Also, the powder tends to pick up water with time. If you store it refrigerated, allow it to come to room temperature before taking some out to weigh.

TEMED can go bad over time. It is moisture sensitive. You use so little to make a gel that it may go bad before being used up. Also, with regards to adding more, I heard somewhere, long ago, that TEMED is the chain initiator, so I would not change the amount. I think it would change the gel polymer.

We had problems with polymerization when we made up "master" gel solutions. Do not premix the acrylamide with the buffer and store it as a stock. It works for a while but then fails. Acrylamide and bis acrylamide can be stored in water, but not with buffer.

Are you new to pouring gels, or is this a new problem? Immediately after pouring the separating gel, you must overlay with water or butanol to exclude the oxygen that inhibits polymerization. If you are using water, you must layer it on gently from one end so as to limit mixing. Also, we used to to put the gel solution (before adding any SDS) in a vacuum flask and attach to a vacuum to reduce dissolved oxygen. The stacking gel should have more ammonium sulfate, and also has the "comb" so it polymerizes without the water overlay.

I think, your protein is of very high MW, pls check. In that case, one option is you can digest it with SDS -buffer -mercaptoethanol for more time, maybe 20-30 mins. and then load into gel.

try a precast gel, and if this does not work, check the generator

Budak. Did u solved ur problem..I am facing some how similar problem. Kindly share your ideas. Thanks

Try using the required min quantity of TEMED and Ammonium persulfate while casting your gels and this may work

Did you connect the terminals correctly to the power pack... ??

Hi Budak,

Hope you solved the problem. If not, check the pH of your Tris buffers as it is critical for the protein migration in resolving. If you solved the problem, could you please share.

Thanks

Priya

With the blood sample from human, my colleague has the similar problem, but for him, protein marker has no problem to enter the separating gel so far.

Hi Wei, Is it because the % of the gel is high?. What percentage gel is he using?

Hi Swapna, 4% stacking gel and 12% separating gel. pretty regular I think.