I had followed the instructions and made 6-15% gradient gel using this above demonstration. To my surprise, it worked and I can easily separe the protein molecular marker ranging from 250-10 KD distinctly. Thanks for uploading the video.
Thank you for this instructional video. Although quite skeptical at first at how the gel would actually run, I was pleasantly surprised at how well the lanes ran and separated. We calculated that pouring a gel costs us a little under $1 AUD so in comparison to a $10-15 gel we will end up saving quite a lot over the lifetime of the lab. Not to mention that having access to whatever percentage gradient gel (3-12/4-16/4-20/10-20) that is best suited for the experiment, is an obvious advantage. Thanks again
I saw this video (after buying a $400 gradient maker) and thought "There's no way in hell this could possibly work". Regardless, I tried it, and color me shocked, it actually works. For those skeptics watching this, give it a try! You won't be disappointed.
Hey don't know if you need it anymore.. However, the gel he is pouring is the resolving gel, the one responsible for the separation of proteins, the one where you insert the comb is the staking gel, which is done and poured after the resolving is completely polymerized Hope it helps
The gels preparation protocol: for two 1.5mm spacers gels I prepare 8ml of each concentration as followed: - 2ml of 4X running buffer (1.5M Tris pH=8.8 (with HCl) and 0.4% SDS, kept at room temp). - Xml of 30% Acrylanide:Bis solution 37.5:1 (make or buy, Bio-RAD cat# 161-0158, or any other company), calculate volume to bring to the desired concentrations. - bring to 8ml with H2O. - 60 microL Ammonium Persulfate 10% (freshly made!) - 1.9 microL TEMED VORTEX WELL before mixing in the pipette. Leave about 15mm for stacking gel and cover with water or butanol. Stacking for two gels : - 1.5ml 4X Stacking buffer (0.5M tris pH=6.8 (with HCl), 0.4% SDS) - 0.6-0.8 ml 30% Acrylanide:Bis solution (3-4%) - bring to 6ml with H2O. - 40 microL APS - 4 microL TEMED VORTEX WELL! YOU ARE ALL WELCOME :^).
Hi I tried today this protocol It seems that the proteins were ran very well. But in transfer process for a nitrocelulose membrane the samples were not transfered. I used 100V in 1 hour in wet system. How do you transfer your samples using this gradient gel? Thank you so much por attention.
Other question: I have to quantify the FASN (270kDa), do you have any sugestion that I can start to detect it with this gel? I thought in run for 2 hour and half with 120Volts. Do you think this is nice or other way? I thank you so much for attention. Hugs.
Do you have other (small) proteins you want to see in the gel? if so, what size? I use to run gels like these to see RyR1 (560 kDa) and FKBP (12kDa) and see them n the same 5-15% gel (doi:10.1038/nature13950 ext. data figure 1). you can design whatever gradient you like based on the sizes of the proteins you want to see. I would recommend running on one of the lanes a prestained protein ladder and follow it as you run.
Awesome! And may I ask does this guarantee that the gel is homogenous across all lanes? I tested this method by adding two dyes separately into the higher and lower percentage gels and observed a not-so-even mixing after pipetting them into the cassette. (Cuz I am afraid that this would undermine homogenous protein transfer out of the gel across lanes at the same molecular weight level)
I know this is a late reply, but i've run into this issue too. Watching the video, it looks like i'm mixing less (not going back and forth across the gel), and my gels set up incredibly quickly. I'm thinking slower polymerization and more mixing would result in greater homogeneity across the lanes.
Dear Tal Lerberbaum, my name is Alisson Rocha, i'm from Universidade de São Paulo - Brazil. I found very interesting the way you do gradient gel. I'd like know that its possible you give me your gel recipe for both percentage. Many thanks for attention, i hope you return. Best regards, Alisson Rocha.
Hello Tal: Could you please share the protocol for the percentages of your gel? I am having trouble separating my proteins and I think this would really help. Thank you!
without, it's the same as a normal western blot protocol just that there's a gradient change in the concentration of the acrylamide of the gel resolving fraction of the gel.
I used this method and it worked really well, thank you!! By any chance has this technique been described in published research or do you use a gradient maker when doing gels for publication?
Tal its a great technique skill! Here in Brasil we call this a "macete" ! :) Thank you for upload this video, and thank to all people here that coment! Are you taking the same volume of the two gel solutions in the pipette? What is the aspirated volume of each solution?
+João Gabriel Ribeiro ; for a 1.5mm spacer ~3.5ml of each solution, you can use identical volumes or if you are worried that the low MW proteins will fall off you can start with a 1ml of the high concentration followed by the gradient or end with a 1ml of the low concentration for more high MW proteins.
I had followed the instructions and made 6-15% gradient gel using this above demonstration. To my surprise, it worked and I can easily separe the protein molecular marker ranging from 250-10 KD distinctly. Thanks for uploading the video.
can u please share the stepwise procedure you followed to 6-15% gradient gel. This is a kind request.
I've been following your protocol for 5 years, and still amazed me how beautifully works. Thank you for sharing it with us. A really life saver.
This is amazing, just made 4-20% native gel with your protocol and it was absolutely perfect, it is now my default protocol . Thank you!!
Could you kindly share your protocol here? Thanks.
Is the 4% for the stacking gel or part of the resolving gel?
@@remia5 you don't necessarily need a stacking gel if you're using a gradient gel.
Thank you for this instructional video. Although quite skeptical at first at how the gel would actually run, I was pleasantly surprised at how well the lanes ran and separated. We calculated that pouring a gel costs us a little under $1 AUD so in comparison to a $10-15 gel we will end up saving quite a lot over the lifetime of the lab. Not to mention that having access to whatever percentage gradient gel (3-12/4-16/4-20/10-20) that is best suited for the experiment, is an obvious advantage. Thanks again
I saw this video (after buying a $400 gradient maker) and thought "There's no way in hell this could possibly work". Regardless, I tried it, and color me shocked, it actually works. For those skeptics watching this, give it a try! You won't be disappointed.
Awesome job, Ran! Always helping out other scientists! :D
This is the best. I did it two days ago and it worked perfectly! Thank you!
You are my HERO !!!!!
Hi! This is a great video. Thanks! When do insert the comb? Or do you make an additional stacking gel separately?
Hey don't know if you need it anymore..
However, the gel he is pouring is the resolving gel, the one responsible for the separation of proteins, the one where you insert the comb is the staking gel, which is done and poured after the resolving is completely polymerized
Hope it helps
when would you insert the comb?
PERFECT-WORKED GREAT-THANK YOU!
incredible! it actually worked very well!
The gels preparation protocol:
for two 1.5mm spacers gels I prepare 8ml of each concentration as followed:
- 2ml of 4X running buffer (1.5M Tris pH=8.8 (with HCl) and 0.4% SDS, kept at room temp).
- Xml of 30% Acrylanide:Bis solution 37.5:1 (make or buy, Bio-RAD cat# 161-0158, or any other company), calculate volume to bring to the desired concentrations.
- bring to 8ml with H2O.
- 60 microL Ammonium Persulfate 10% (freshly made!)
- 1.9 microL TEMED
VORTEX WELL before mixing in the pipette.
Leave about 15mm for stacking gel and cover with water or butanol.
Stacking for two gels :
- 1.5ml 4X Stacking buffer (0.5M tris pH=6.8 (with HCl), 0.4% SDS)
- 0.6-0.8 ml 30% Acrylanide:Bis solution (3-4%)
- bring to 6ml with H2O.
- 40 microL APS
- 4 microL TEMED
VORTEX WELL!
YOU ARE ALL WELCOME :^).
Hi
I tried today this protocol
It seems that the proteins were ran very well.
But in transfer process for a nitrocelulose membrane the samples were not transfered. I used 100V in 1 hour in wet system.
How do you transfer your samples using this gradient gel?
Thank you so much por attention.
Same as any other gel: Tris-glycine buffer with 20% methanol, I typically use constant amperage at 200mA for an hour in a chilled system. HTH.
Other question: I have to quantify the FASN (270kDa), do you have any sugestion that I can start to detect it with this gel?
I thought in run for 2 hour and half with 120Volts. Do you think this is nice or other way?
I thank you so much for attention.
Hugs.
Do you have other (small) proteins you want to see in the gel? if so, what size? I use to run gels like these to see RyR1 (560 kDa) and FKBP (12kDa) and see them n the same 5-15% gel (doi:10.1038/nature13950 ext. data figure 1). you can design whatever gradient you like based on the sizes of the proteins you want to see. I would recommend running on one of the lanes a prestained protein ladder and follow it as you run.
The other protein is the Beta Actin (43kDa).
Gorgeous!😃
its really a good idea. Is anyone worked on a gel with 2 different properties varying in 2 dimensions ?
Buddy you are an angel to me 🙇
Awesome! And may I ask does this guarantee that the gel is homogenous across all lanes? I tested this method by adding two dyes separately into the higher and lower percentage gels and observed a not-so-even mixing after pipetting them into the cassette. (Cuz I am afraid that this would undermine homogenous protein transfer out of the gel across lanes at the same molecular weight level)
Did it gave a homogeneous result after polymerization? It might need a bit of time to settle, but that's only a guess.
I know this is a late reply, but i've run into this issue too. Watching the video, it looks like i'm mixing less (not going back and forth across the gel), and my gels set up incredibly quickly. I'm thinking slower polymerization and more mixing would result in greater homogeneity across the lanes.
It's amazing thank you
Dear Tal Lerberbaum, my name is Alisson Rocha, i'm from Universidade de São Paulo - Brazil. I found very interesting the way you do gradient gel. I'd like know that its possible you give me your gel recipe for both percentage. Many thanks for attention, i hope you return. Best regards, Alisson Rocha.
Thank you very much
Hello Tal:
Could you please share the protocol for the percentages of your gel? I am having trouble separating my proteins and I think this would really help. Thank you!
What voltage and for how much time do you transfer the gradient gel to membrane?
Nice Skill!
Do i have to make stacking gel also, for gradient gel?
+freesoul YES.
For gradient gels, do you use transfer buffer with or without SDS?
without, it's the same as a normal western blot protocol just that there's a gradient change in the concentration of the acrylamide of the gel resolving fraction of the gel.
I used this method and it worked really well, thank you!! By any chance has this technique been described in published research or do you use a gradient maker when doing gels for publication?
+Betty haha, no this is the only publication for the method, guess you can cite this video :^)
Any updates on the citation for this method?
Tal its a great technique skill! Here in Brasil we call this a "macete" ! :) Thank you for upload this video, and thank to all people here that coment! Are you taking the same volume of the two gel solutions in the pipette? What is the aspirated volume of each solution?
+João Gabriel Ribeiro ; for a 1.5mm spacer ~3.5ml of each solution, you can use identical volumes or if you are worried that the low MW proteins will fall off you can start with a 1ml of the high concentration followed by the gradient or end with a 1ml of the low concentration for more high MW proteins.
Чёт какая-то хрень, ну закажи у токаря градиентом, чё мучаться.