How to make gradient gel for western blot ? - (Oct/11/2008 )

I buy precast 4-12% Gradient Gel but it cost quite a lot.
Is there any way I can do by myself ?

---

Yes, you can. To make a gradient gel you will need a 'Gradient Gel Casting Chamber'. However I have not actually used one before and can not tell if it is easy to use such a devise.

Gradient Gel Caster

---

as perneseblue says; you need a gradient mixer which fits to your gels system; you have exactly to fill the correct volume (= volume of total separating gel) in the gradient mixer tubes; the higher concentration (f.i. 12%) is near the drain tube; constant mixing is necessary;


you certainly will need some attempts to succeed; as the reproducibility of these gels is difficult, we also use precast gels

---

These gels are not impossible, but are more complicated to make than normal gels.

I assume you have a good reason for running gradient gels (you want to detect more than one protein and they run close to each other). If you have made this choice, you need a gradient maker.
You can fashion one yourself (or buy), its basically two tubes, connected at the bottom and with an exit tube. Valves are fitted between tubes and you need a small magnetic stirred at the bottom of tube 1. Tube 1 gets the highest percentage of acrilamide and its the tube with entry and exit connectors, tube 2 gets the lower. Your temed concentration is normal, but Amonium persulfate will have to be adjusted (5 to 10 times less). After you have prepared your acrilamide, you open the valve on 1 and start pouring from the highest concentration. With your stirrer going as fast as possible, you open the connection between tube 2 and 1. Because you have already poured from tube 1, the pressure from tube 2 will force a continuous dilution of the solution at the exit point. The gel will polymerize slowly. At this point, you need to ask yourself: Do I need gradient gels?

Best regards

Alejandro

---

If you are talking about Mini-Gels, I would not try to cast them by yourself, it is possible in a larger format, but the procedure is not so easy and the gradient maker is also costly. Only if you need them for a long time and won't buy them all the time it is worth to learn the procedure and buy the things. Otherwise try to get a good discount

---

buy precast gel...expensive but give consistent results

---

Thank you very for all valuable comments!

yes, I'm using Mini-Gel to check proteins from my friends who have done from 2D gel.
There are 3 proteins which MW really close each other and the precast gradient gel give me very nice result.

According from everyone comments, it seems that precast gel is better way. If it sound impossible to do by myself, I may buy precast gel then.

Thanks,
/Tai

---

not impossible but needs some skill; in my old lab we have done it routinely even for mini gels...

---

I prepared 4-20% gradient gels on Hoeffer 8 cm gels and 10-20% gels on Hoeffer 10 cm gels for years. The other benefit to doing these yourself, outside of them being more cost effective, is that the gels are gorgeous looking when done properly. Knowing some of the tips going in makes it much easier. It is totally doable! You will for sure need a gradient maker, one that holds 50 ml max (ballpark) in each chamber. Look around your institute for an old one - someone out there has one collecting dust. I can help you more with a protocol and setup if you like.

Go for it!

---