6 replies to this topic

[Genecks]

Hello,

Something I have been trying to figure out for a while is what the percentage means when talking about various percentages of SDS page gels. When I read a protocol saying I need to add 1% triton x-100 or 0.5% NP-40 or something like that, I understand that my final solution needs to have that particular solution as a percentage of the solution.

However, I have not been able to figure out how this relates to the SDS page gels. I have looked over various protocols, and I can't see what compound, chemical, or solution allows the gel to be called a particular percentage.

What does percentage mean when dealing with stacking and running gel for SDS page?

In a lot of ways, I'm trying to figure out why particular amounts of chemicals are used.
For instance, I use a 7.5% separating gel recipe of lesser volume, but the proportions used are similar to those calculated on this website:
http://www.cytograph...lab/acryl3.html

So, I'm trying to figure out why these calculations work out this way. Where are these calculations coming from?

Edited by Genecks, 05 May 2012 - 08:28 PM.

[bob1]

The percentage is the percent acrylamide in the solution.  If you want a 7.5% acrylamide gel from 30% starting solution and 10 ml final volume use:

C1xV1=C2xV2
7.5 x 10 =30 x v2
V2=75/30
V2=2.5 ml  Therefore add 2.5 ml of 30% acrylamide solution to a tube, add the other components to a final volume of 10 ml

[Genecks]

bob1, on 06 May 2012 - 01:46 PM, said:

The percentage is the percent acrylamide in the solution.  If you want a 7.5% acrylamide gel from 30% starting solution and 10 ml final volume use:

C1xV1=C2xV2
7.5 x 10 =30 x v2
V2=75/30
V2=2.5 ml  Therefore add 2.5 ml of 30% acrylamide solution to a tube, add the other components to a final volume of 10 ml

How would I know what the volume and concentration of the other components should be to make a successful gel? It seems like for the recipe, the acrylamide is the independent variable, and the other components are variable that depend on the acrylamide concentration added to the running/stacking solutions.

Edited by Genecks, 07 May 2012 - 12:04 AM.

[alan.hargreaves@ntu.ac.uk]

The other comments contain clear advice that addresses your original question.

The acrylamide concentration can be varied to achieve good resolution for different molecular weight ranges. You can purchase a pre-made soultuion of acrylamide (which is a mixture of acrylamide and bis acrylamide, a cross linking agent). The total volume in any gel recipe is constant, which is achieved by changing the amount of distilled water added to make up the final volume.

Therefore the concentrations of other reagents remain constant. It seems to me that you don't have  a protocol. If you purchase some acrylamide/bis acrylamide solution the supplier will probably send a datasheet with suggested volumes of all reagents. Alternatively protocols are available online and can be readily found using Google.

If your knowledge of protein separation techniques is very basic, I would suggest reading a Practical Techniques text book on SDS-PAGE (try Amazon.com). In addition to providing theoretical knowledg about SDS-PAGE, this would also contain protocols for casting gels.

[Genecks]

Well, I do have a protocol that i'm working from.
What varies between them is the amount of solution used, particularly the amount of ddH2O used.

bottle of acrylamide/bis: http://www.bio-rad.c...de/Bis_Solution

Quote

7.5% gel (20.015 mL)

------------
10% AP| 200 ul
Monomer solution (30% acrylide/bis (29:1))| 5 ml
4x running buffer| 5 ml
10% SDS| 200 ul
ddH2O| 9.6 mL
TEMED| 15 ul

12.5% gel (10.01 mL)
-------------

10% AP| 100 ul
Monomer solution (30% acrylide/bis (29:1))| 4.15 mL
4x running buffer| 2.5 ml
10% SDS| 100 ul
ddH2O| 3.15 mL
TEMED| 10 ul

So, with this protocol, i don't see where the variables are changing, why, and how.

alan.hargreaves@ntu.ac.uk, on 07 May 2012 - 09:49 AM, said:

The other comments contain clear advice that addresses your original question.

The acrylamide concentration can be varied to achieve good resolution for different molecular weight ranges. You can purchase a pre-made soultuion of acrylamide (which is a mixture of acrylamide and bis acrylamide, a cross linking agent). The total volume in any gel recipe is constant, which is achieved by changing the amount of distilled water added to make up the final volume.

Therefore the concentrations of other reagents remain constant. It seems to me that you don't have  a protocol. If you purchase some acrylamide/bis acrylamide solution the supplier will probably send a datasheet with suggested volumes of all reagents. Alternatively protocols are available online and can be readily found using Google.

If your knowledge of protein separation techniques is very basic, I would suggest reading a Practical Techniques text book on SDS-PAGE (try Amazon.com). In addition to providing theoretical knowledg about SDS-PAGE, this would also contain protocols for casting gels.

I took a look and noticed BIO-RAD has a nice document about their product. I suspect the formulas provided are what I would be using to determine percentage, right?

http://www.bio-rad.c...lletin_9413.pdf

I wonder what I would do if I did not have the BIORAD solution, however. I have to make some solutions from powder as of late.

Edited by Genecks, 07 May 2012 - 05:56 PM.

[Genecks]

Ok, I made a spreadsheet now that I know the math. This spreadsheet is for the running gel solution. What strikes me is the amount of TEMED and Ammonium persulfate used. In my protocol, it seems like having at least 10 uL for a 10 mL 12.5% separating gel solution should be used. But my calculations provide otherwise.

Anyway, here is a contribution ( percentage spreadsheet for 30% Acrylamide/Bis Solution #161-0156 ):

Attached Files

•  SDS%20Page%20Percentage.xls   8K   112 downloads

Edited by Genecks, 07 May 2012 - 05:58 PM.

[bob1]

Genecks, on 07 May 2012 - 04:18 PM, said:

Well, I do have a protocol that i'm working from.
What varies between them is the amount of solution used, particularly the amount of ddH2O used.

Quote

7.5% gel (20.015 mL)
------------
10% AP| 200 ul
Monomer solution (30% acrylide/bis (29:1))| 5 ml
4x running buffer| 5 ml
10% SDS| 200 ul
ddH2O| 9.6 mL
TEMED| 15 ul

12.5% gel (10.01 mL)
-------------
10% AP| 100 ul
Monomer solution (30% acrylide/bis (29:1))| 4.15 mL
4x running buffer| 2.5 ml
10% SDS| 100 ul
ddH2O| 3.15 mL
TEMED| 10 ul

So, with this protocol, i don't see where the variables are changing, why, and how.

Note that the total volumes for these two solutions are different - multiply the 12.5% gel by 2 to get the same final volume as the 7.5%...

Genecks, on 07 May 2012 - 04:18 PM, said:

I took a look and noticed BIO-RAD has a nice document about their product. I suspect the formulas provided are what I would be using to determine percentage, right?
http://www.bio-rad.c...lletin_9413.pdf
I wonder what I would do if I did not have the BIORAD solution, however. I have to make some solutions from powder as of late.

If you didn't have the biorad solutions, you can easily make solutions from powder to be the same as the biorad ones... these are all standard protocols for acrylamide gels.  I suggest you find a copy of "Molecular cloning: a laboratory manual" by Sambrook et al., and have a look through that.

Genecks, on 07 May 2012 - 05:10 PM, said:

Ok, I made a spreadsheet now that I know the math. This spreadsheet is for the running gel solution. What strikes me is the amount of TEMED and Ammonium persulfate used. In my protocol, it seems like having at least 10 uL for a 10 mL 12.5% separating gel solution should be used. But my calculations provide otherwise.

Not that your maths is particularly wrong, but there are easier ways of calculating the volumes of dilutions for the percentage gels.  The volume doesn't need to be too exact; 0.05 ml is well within the margin of error for measuring 100 ml, so your proportion aspect should be 0.1 not 0.0999...

If you really want to know how the volumes/concentrations are derived, I will refer you to Ornstein, 1964,  Ann N.Y. acad sci, 121:321-349, Davis, 1964, Ann N.Y. acad sci, 121: 404-427 and Laemmli 1970 (one of the most cited papers ever, you should have no problem finding it).