Most of these questions should be addressed in the user's manual that came with the SDS-PAGE electrophoresis apparatus, or you could consult Maniatis. But briefly, I will share that I layer water-saturated butanol on top of my separating gel to prevent contact of the polymerizing gel with the air. This makes for a cleaner interface. Also, polyacrylamide mixes (usually the 30%/0.8% acrylamide/bis solution) can be purchased as liquids, are kept in a brown bottle at 4C for storage. 10% APS is best prepared fresh, but can be stored at 4C. However, it may lose its potency if stored for longer than 1-2 weeks.

Prestained protein markers typically don't require heat denaturation prior to loading on the gel, as they are already in denaturing/reducing loading buffer.

Finally, your choice of setting on your power supply can depend on the gel and what MW range proteins you are trying to separate. Typically, for a 10% thick (1.5-mm) SDS-PAGE, I would apply 125V (constant voltage) for about 1-2 hours.

Hope this gets you started, Budak. But don't be afraid to read the manuals and product literature for directions. And it helps to ask someone in the lab (usually a helpful postdoc) for a little instruction.

On top of the resolving gel you should add water, it will prevent interface with water and keep your gel straight without any air bubbles. You can prepare a 1 ml stock of 10% APS, aliqote it and keep it in the -20 degrees, don't thaw and freez. In case your gel still doesn't solodify try to add 1-2 ul of TEMED, it should do the trick.

Thanks.

There is a similar problem encountered previously in my old lab. It might be the problem with the APS. APS salt crystals should be stored in the cold and sealed tight when not in use. If the APS is functioning right, the addition of water to the salt will result in some cackling sounds. If not you might want to try to get a new bottle of APS

So, APS is the most important solution for preparing SDS-page. But, why it will result in cackling sound after adding water?Is it any specific reaction during the process?

APS is a highly reactive oxidizing agent. I suppose that is the reason to the cackling. Though I might be wrong.

Is it recommended to degas the gel mixture before adding APS and TEMED? Oxygen is the crucial thing to be avoided of inhibiting the polymerization process of gel. But, If APS works well, will the gel get solidified after pouring between the plates in closed casting frame although the oxygen is present and the bubbles formed in gel mixture?

can I use 95% ethanol to layer on top of separating gel instead of using isopropanol?But, Is water recommended to layer on top of separating gel?

Can I prepare for aliquot of 10% APS in 1.5 ml centrifuge tube and stored at -20c freezer?

What is the recommended percentage of iso-propanol for that purpose?

Usually if your SDS-PAGE resolving (separating) gel does not solidify, it has something to do with the Ammonium Persulfate (APS) and TEMED. I usually mix water, poly acrylamide (1:29), and the resolving gel buffer first. Then, I add 10% APS and TEMED quickly, mix, and add to the gel caster. Sometimes if the APS is not fresh, or diluted, the gel will not polymerize. Hope this helps.

I found that it is best to make APS fresh each time you make a new gel.

Thanks Darius. Are you graduate student from Georgia State University?

You're welcome! No, I am a senior undergraduate. I have been in the lab for 3 years now trying to learn as much as I can

From your point of view, which part of biotechnology you like most?

I like protein expression and purification. I enjoy gene cloning too but it is much more difficult, at least from my experience. What about you?

You get much better and consistent results if you simply buy premade gels. When you factor in the cost of your time in preparing your own gels then you are actually wasting money.

Other very great option is to add glicerol to the separating gel. I use to add 1mL of 80% Glicerol (and 1mL less of water). Then, you can directly apply the stacking gel on top without waiting. They won't mix with each other since the separating gel will be more dense due to the glicerol. Very useful!!

I agree with Alison Winger - she provided a clear and concise explanation - only water is needed on the top - and making up the APS fresh each time you use it or aliquoting it and freezing it works as well. Good luck!

Dear Budak

For bigger gels (17 cm and above) you must to use propanol or other alcohols on the surface of gels but in smaller gels (11 and 7 cm) you can use only D.W.

I always keep all of SDS-PAGE reagent except SDS 10% in refrigrator. Between those, only TEMED and Acryamid/Bisacrylamid need to be kept in dark bottle.

Proetein marker just keep in -20 C freezer.

APS is better to prepare at days of gel running. However you can use it for 1 week after preparation.

I use with two parameter in different works. For SDS-PAGE I use constant current but in 2DE I used constant voltage. Keep in your mind that constant current is important than constant voltage.

For not solifying of your gel:

1st: Please check APS and TEMED for expiration and keeping condition.

2nd: Please check the temperature of your laboratory.

3rd: You can increase the volume of TEMED and APS.

I run several 1DE and 2DE gels and have complete protocols of those. If you have problem yet, please dont hesitate to contact me.

Cheers

Dear Budak,

It is indeed necessary to insulate the top layer of the resolving/separating gel with a liquid to prevent the loss of free radicals and generate a smooth upper layer of the separating/resolving gel. In general, water can be used for this purpose, although it is better to use butanol, if possible. Next, I would also suggest you to keep the gel apparatus at 37 degrees for at least 20 minutes before checking for gel solidification.

As far the storage properties of the solutions are considered,

1. The solution of Acrylamide - BisAcrylamide should be kept in a brown colored bottle at 4 degrees.

2. SDS solution should be kept at room temperature (Do NOT store this in the refrigerator!).

3. TEMED should be stored in a brown colored bottle at 4 degrees.

4.Ammonium persulphate solution should always be prepared fresh as required and should be stored at 4 degrees, till usage.

5. All the buffers can be stored in a transparent bottle at 4 degrees.

The answer to your first question is YES! You can directly load the promega protein M.W. ladder after thawing it completely. However, it is appropriate and useful that the samples should be heated with SDS at 100 degrees for at least a couple of minutes (You can SKIP this step if you work with an intrinsically unstructured protein).

I am not sure of the answer to your second question since, the separation depends on more than one factor and the conditions therefore, would vary according to the resolution that you want to achieve.

You can get answer for all questions by a simple search on the Internet. Anyway, the solution you have to maker fresh is ammonium persulfate just before making the gel. Layering of serating gel with water added carefully will make the gel surface even.

Dear Melayu,

Your problem is very common one. You need to check out some steps.

First i would like to tell you about the storage of different components of SDS-PAGE

1) 30% bis-Acrylamide (29% of acrylamide and 1% of bis-acrylamide) store in dark bottle and in 4 degree centrigate.

2) Tris buffer (1.5 M) Ph 8.8 and Tris (1M) pH-6.8 these should be in room temperature.

3)10% SDS same as Tris buffer in room temp.

4)APS(10%) Should be freshly prepared or not old then 2-3 weeks this you keep in 4 degree.and keep in dark container because its light sensitive {APS &TEMED is the most important composition for solidifying the gel}

5) TEMED is also light sensitive so should be in dark and 4 degree..

Regarding your problem make fresh APS(10%) caste the gel..after pouring the resolving gel just give few ul of (around 300-500ul) of isopropanol above the gel.It makes the layer straight and helps in solidifying the gel too..

As many places its written that APS should be fresh but in our lab we generally prepare 10-15 gels per day and we use APS till 2-3 weeks old. There is no problem about it.

So check out all the composition properly and try to caste it..

hope you will be successful..all the very best.

Dear Gaurav

Thanks for your comments

I use APS after 1 month without any problems too.

According to my knowledge, solidifying of gels are not related to the nature of liquids that added to the surface of the gels. These liquids are just for equilibration of the gel surface and avoiding from air penetration.

In the original protocol of Laemmli Tris buffer 0.5 M was used for stacking gel but you use 1 M. Please cite your reference(s).

Sincerely

Is it necessary to put any liquid on top of TEMED-treated separating gel mixture between spacer plates with 1.0 mm Integrated spacers in closed casting frame?If yes, what type of liquid (water,miscible, immiscible or partially miscible liquid) is proper to use?

ANSWER: It is mandate when you are preparing the gradient gel or leaving the separating gel for long time other wise you may left it as for polymerization which need 15 to 30 minutes.

For not solidified gel, please give me general guide on troubleshooting it because i am truly novice with this SDS-page work.

ANSWER: you may find troubleshooting on internet in current protocols.

Besides that, which solutions of preparing SDS-page need to be kept in:

a)dark-brown bottle:

acrylamide+bis-acrylamide solution, APS powder, TEMED.

b)room temperature:

tank/ electrode buffer, gel buffer, SDS solution

c)chiller at 4 degree celcius:

sample buffer/ gel loading dye, TEMED, APS powder.

d) minus 20 degree celcius freezer:

APS solution max upto 1 month, protein marker, protein/enzyme samples.

And, also which solution is proper to prepare fresh on the day of performing SDS-page.

ANSWER: If not storing at -20 than APS solution should be prepared immediately when required. (this is recommended then stored APS solution)

*Can I use unstained broad range protein marker (promega) directly to apply in SDS-PAGE without incubating it for a while together with protein samples at 100 degree celcius in water bath/ heat block?

ANSWER: here you need to follow manufacturer instruction. better to give same treatment.

*The last question is, which condition is better for separating protein sample in terms of different size of protein bands, either using constant voltage or constant current, included also the parameter of power (watt)?

ANSWER: constant current is most recommended and also check reproducibility of power (watt) at every repeat of same experiment. if there is any change in voltage and watt at same constant current then check the strength and pH of tank and gel buffer (recommended use fresh tank buffer after 3 to 4 gel run and fresh gel buffer after one month/ appearance of contamination.

You should keep 10% APS solution at 4 and no longer than one mounth

Dear Carolin

I and other researchers used 10% APS. The volume that I used and cited in references is 50 ul. Can I prepare APS with higher concentration and use smaller amount?

I never do that until now.

If other people have suggestions, please discuse about them.

In response to dear hardev

You have three choices when preparing the gels:

1. You can make gels, and seal it with parafilm and store it for 24-36 hours in4 C.

2. You can prepare the separating gel and wait to solidify and then added stacking gel.

3. Also in other way that I prefered and do that more time, I pour the seperating gels and immidiatly add stacking gel and comb. Because of different concentrations, the gels are good polymerized and ready to use after15-20 min.

APS should be freshly prepared everytime..

Dear Masood, Sure! you can use 20% APS as well I usually do it and it works well!

Normally conservation at 4 for one mounth is OK for both concentration!

Thanks a lot Dear Caroline

What is your using volume of 20% APS to prepare 12% separating gel?

I use the same volume of APS and Temed for any % of gel.

For a 10ml running solution I use 20 µl APS 20% and 5 µl Temed.

For a 10ml stacking solution (for 2 baby gels) I use 75 µl APS 20% and 15 µl Temed

Dear Caroline

I usualy use 50 ul APS 10% and 5 ul TEMED (Ratio 10:1) for 10 ml of all percentage of both separating or stcking gel according to Laemmli, 1970.

According to your response, this ratio is 8:1 for separating gels and 10:1 for stacking gels.

Also, it seem that the volume that you have used for stacking gels is too high.

Please cite the references that you extract this protocole from that, if possible.

Sincerely

It's a protocole used in my lab for 10 years and in my previous lab as well!

And sometimes when i don't have the time to wait too long polymerisation I double the concentration of each APS and temed! AND it's works as well!

High concentration of APS and TEMED will fasten the polymeraization of the gel but high concentration of these chemical may interfere with the mobility of the protein/ charge molecule.

for this you should run the gel for about 15 to 30 minutes before loading the samples as pre-run to remove such molecules.

Hello,

You can freeze APS at -20°C to preserve it. You make some aliquot of your APS with the volume that you use usually and so you thaw it before you prepare your gels.

It works very well!

For your problem of no polymerization, be sure that your APS and TEMED are good (not expired) and that the room temperature is not too low (if t°C is low the polymerization takes more time than in higher t°C).

You can see in my articles if you want to see the protocoles used for SDS-PAGE.

Best regards.

Hi

I suggest you please check your buffer besides concentration of APS or TEMED. I also had a problem of no polymerization of resolving gel but it was solved only after changing the buffer. The high concentrations of APS or TEMED did not help, neither low temperature.

U check and try it again. Hope it will work

Best Regards

APS should be freshly made. TEMED also gets bad with time. Sometimes storing the gel in the 37oC incubator helps the stacking to polymerize. Or increase the percentage of acryamide in the formulation.

Try to avoid using buffers (pH specific) that are over a month old. Prepare APS immediately before use, I would not bother freezing it. This has made a big difference for me in the past. If this does not help, check your TEMED, it usually lasts for a very long time, but it might be very old?!

Check the APS and TEMED

Polymerization is affected by many factors. Make sure your precursor solution is degassed, at room temperature. Detergents and extreme pH's can also inhibit normal polymerization. Check your initiators. Having too much surface exposed to air can also inhibit polymerization. This is a great reference to understanding the PAG polymerization process. The more you understand, the easier to troubleshoot! www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_1156.pdf

hello,

90% of polymerization problems come from the APS. so make sure the following steps:

it's so much better using a fresh APS (10%) (1g of ammonium per-sulfate resuspended in 10 ml dH2O) (Ammonuim per-sulfate should be in the fridge) mix slowly, aliquote into small tubes (300 ul) and keep them in the freezer.

for each experience try to take only 1 tube and keep the rest for long time.

if the problem still there check your TEMED.

ENJOY 100% SUCCESSFUL WORK.

I encountered similar problem, the left out gel mixture polymerized in the tube ,but the resolving gel in between the spacer plates was not polymerizing till long time, At last i discarded and made a new gel.Sometimes i came across this problem when TEMED gel mixture could not polymerize between the spacer plates , but left out resolving gel in tube polymerizes quickly. If anyone has answer for this ?

the bis acrylamide may be expired

TEMED and APS are the factors interfere with polymerization. APS should be prepared freshly . TEMED used for PAGE unexpired.

From my personal observation, the culprit is APS, any time I used left over or old APS, my gel especially the lower gel doesn't polymerize but when I prepared APS fresh, It polymerizes within thirty minutes maximum.