Do you buy your gels and buffers or make your own? Just be sure you don't miss anything. I am not quite sure why the gel stopped running? Maybe you want to check your power connection to make sure it is properly connected. Then run some other protein samples just to be sure your gel is running as it's supposed to be.

Hi Supriya, Hua asked the definitive question. Do you use commercial gels and buffers or do you make your own?

Hua Xiao Steingrimur Stefansson We make our own gels and buffers. It runs well for other samples. I have encountered this problem only with this human serum.

I did have a problem with the voltmeter, so I had to change it to another source. But the bubble stopped after changing the setting.

hi Supriya,

please check these:1-leakage of tank,2-pH of gel and running buffer, 3- voltage

Hi Supriya, the gel looks like it hardly ran at all.

What does the bromophenol blue dye front look like, sharp or diffuse? If it is diffuse then your gel rig might be leaking like Mohammadbagher mentioned.

1. The running buffer is often prepared as 10x and has to be diluted before use - did you do this?

2. Do you have an idea about total protein concentration in your sample and have you varied the concentration?

3. What kind of buffer is the sample in? High concentration of salts or urea might disturb the run. Did you boil it before the run and does the sample buffer contain DTT?

4. Does the glas plates become hot during the first minutes of the run? Maybe the system is overheated, in that case try to use lower voltage for a slower run.

شكرا

Hi Vivica,

Thank you so much for the response. Here is what i did during this experiment.

1. I used 1x running buffer

2. Yes, I did know the protein concentration and yes I used different concentration for all proteins. I wasn't aware that we need to add same concentration of different samples.

3. My sample has 10% TECP and and an alkylation agent CAA(10%). No urea or DTT

4. I did not notice the glass getting hot, may be because I did not check on it in the first 5mins. I will look into it the next time.

I really appreciate your advice and help here.

Regards.

Hi Supriya, did you use Tris-HCl or Tris base to make the running buffer? Does the gel rig leak?

Is there anybody else having the same problem?

Dear Supriya, by simply taking into account few steps your issue can be sorted easily.

Check the connections of your power supply, they must be tight enough and not rusty.

Gel tank banana plugs should appear very clean.

Run the gel with fresh buffer ( not to be used above 6-7 times, though some recommend to use till 10 times).

Running buffer pH is of course important, as increased alkalinity renders the proteins to move slowly in gel.

Before running the experiment keep the empty gel tank in fridge at 4 C for couple of hours.

Composition of your stacking gel is very crucial for the migration of proteins, be sure there is no mistake at all in this, whether technical mistake or calculations).

Often the DNAse treatment or sonication of protein lysate helps in this situation you are encountering, as it is a known fact that increased nucleic acids in the lysate and the presence of heavily glycosylated proteins result in an enhanced hydrophobic interactions of proteins, thus immobilizing them on gel.

Also, gross overloading of proteins blocks the gel matrix pores and proteins ultimately cannot find their path. Be sure to load reasonable amounts of ug of proteins.

One recommendation I would like to suggest that do a quick spin at 4 C to your protein-sample loading buffer mix, take supernatant carefully, transfer to other tube and then perform heat denaturation step rather doing it all together.

Regards....

I was also having same kind of problem ( overheating, smeared band shape, non linear progression) while running gels with Human serum sample. Glycosylated proteins or some very high molecular proteins in the serum can cause this problem although I couldn't find the solution. Please let me know if you can solve the problem . Thanks .

Where did you get your protocols from? You said you are new to PAGE and Western blot - did you get a theoretical idea about the general principle of the techniques?

10% TCEP and 10% CAA seems incredibly high to me - is there a reason why you use such high concentrations? Especially the high concentration of CAA seems to be prone of unwanted reactions besides alkylation of TCEP-reduced SH-residues.

Typical concentrations of TCEPs are around 5-50mM (around 1%) and typical concentration of of CAA are around 20-50mM (0,2-0,5%).

Do you add TCEP and CAA at the same time of one after the other?

Besides the high concentrations, both components have a low pH in aqueous solutions - are you sure that the pH of you sample buffer is still correct?

I guess your PAGE system is the method of Laemmli. It seems to me that the conditions are not appropriate. What if you recheck the concentrations of Tris, Gly, SDS, Tris-HCl, and pH, i.e. your recipe? For example, 375 mM Tris-HCl, pH8.8 in the separating gel, 125 mM Tris-HCl,pH6.8 in the stacking gel, etc. (Of course, don't forget 0.1% SDS)

Havnt get-it

Guys thank you very much for all your precious replies. It definitely helped me troubleshoot my issues. I was finally able to get the gel up and running, sorry about the late update here. It was a leaky system, I had to replace the cassette and increase the voltage upto 200. It ran perfectly fine.

Thank you all so very much!