LabTricks » How to Stain an SDS-PAGE gel

Date: 23 Feb 2011

Tag: biochemistry, electrophoresis, MBB309, protein, SDS-PAGE, video tutorials

Comment: 11

Your gel just finished running – what do you do next?

In this video, Dan shows you how to disassemble your SDS-PAGE gel, and how to stain it using PageBlue, Coomassie, or Silver Staining.

Coomassie is the older method of staining, and gives bright blue bands on the gel. PageBlue is similar, but provides a quicker alternative. If you have faint bands and require a more sensitive method, you can use Silver Staining.

Choose whatever method you need, but beware of the hazardous materials that may be used.

Watch these videos first:

- How to Make an SDS-PAGE gel
- How to Run an SDS-PAGE gel

Got leaky gels? This video will help:

- How to Avoid a Leaky SDS-PAGE gel

11 Comments

pipit June 26, 2011 at 6:14 pm

Hi.., I am newbie in PAGE. Ive followed every trick that you've served in this web and i adopted some tricks for my work, such as : how to avoid the leaky gel. Fortunately, it work well! Currently, i desperate about avoiding the torn gel. the gel always tear once i remove glasses. It sticks side by side to the two of glasses caster. Ive utilized SE 600 (GE) for my PAGE work. I wonder how to repel the glass without making any damage. Do i need to mind stages before pouring the gel? Should i use the repellent chemicals? If so, would you mind to give me more information.
- labtricks June 26, 2011 at 8:38 pm
  
  Hi pipit, Glad to hear that we've been helpful! One simple trick that I've found very useful (and it was briefly mentioned in the video above) is to separate the glass plates while your gel+plates are either under running water, or placed in a container with water (if you don't want to use water, your running buffer can also be used). The idea is to let the water/buffer get in between the plates, to help the plates come apart. This way you have to use less force yourself, and the gel won't rip. After you have removed one plate, take the remaining plate with the gel still attached, and do the same thing. In the video (at 1:31) we show that if you put the gel+plate into the water/buffer with the gel facing down, the gel slides right off (you can also gently use the wedge tool to help it). Try using this trick and let me know how it goes. Good luck! -Suraaj
ebor September 19, 2011 at 1:31 am

cool......want to see some more cool stuff....!!!!!
Wilmer November 20, 2011 at 2:49 pm

hey, thank you very much for this video!!! just today I was running my first SDS-PAGE gel and I didnt know how to ensamble it... I followed your tips and finally my gel was great!!! Here 2 pics of my SDS_PAGE gel =) http://a2.sphotos.ak.fbcdn.net/hphotos-ak-snc7/s720x720/319992_2499451559860_1060049621_2782684_733348852_n.jpg http://a3.sphotos.ak.fbcdn.net/hphotos-ak-snc7/s720x720/303805_2499450799841_1060049621_2782683_1136119256_n.jpg
Anup March 30, 2012 at 7:44 am

thanks for the information how to make SDS-PAGE.....I have a Question.....Why polyacrylamide gel is high resolving power but small range of separation power??? I am very grateful to you if you answer this....
- labtricks April 9, 2012 at 3:14 pm
  
  Hi Anup, Polyacrylamide is said to have high resolution meaning that it is good at resolving two bands of similar molecular weight. But it is said to have small range of separation because that is dependant on the % gel you make. Based on the amount of acrylamide and bisacrylamide you use, the size of the pores inside the gel will be different, and for a specific range. As you add more acrylamide, the pore size decreases, which is better for smaller proteins. For more detailed explanation, here are some links: http://en.wikipedia.org/wiki/SDS-PAGE#Chemical_ingredients_and_their_roles http://homepages.gac.edu/~cellab/chpts/chpt4/intro4.html Hope that helps! -Suraaj
Lori June 23, 2012 at 11:46 am

That was really cool! Thanks for the post!
Lucy April 5, 2013 at 11:37 pm

What's the difference between staining the SDS-PAGE gel with PageBlue, Coomassie, or Silver Staining verses using a blocking solution, primay antibody, and a secondary antibody? Do I need to stain using PageBlue, Coomassie, or Silver Staining, if I'm going to use an antibody? I'm planning on performing a Western Blot for the first time. Further tips or information, is very much appreciated. Thank you.
- labtricks April 8, 2013 at 10:14 am
  
  Hi Lucy, When you are doing a Western blot, you do not stain the gel that you are blotting. Usually, most people will make and run two identical SDS-PAGE gels. They will stain one gel (with Coomassie, PageBlue or whatever), and the other gel is used directly for the Western blot procedure (without staining). This way, you can use the stained gel as a visual guide and understand which protein band your antibody is binding to. -Suraaj
biochemist April 7, 2013 at 1:50 am

thanks a lot for this video , but i have a question why do we use multiple PH values within a single gel? i will be happy if you answer me.....
- labtricks April 8, 2013 at 10:19 am
  
  Hi, The multiple pH values allow the protein to migrate down the gel. In a standard Laemmli Tris-Glycine system, the pH of the different components is as follows: -Running buffer with its natural pH of about 8.3 (no pHing step is usually required for this buffer). -Resolving/separating gel is a few tenth of a unit higher than running buffer (at pH 8.8). -Stacking gel is about 2 units below running buffer (at pH 6.8). -Sample buffer pH is usually the same as the stacking gel at pH 6.8. The glycine in the running buffer (pH ~8.3) is negatively charged, and will move towards the anode (positive), entering the stacking gel and sample. Here the pH is 6.8, protonating the glycine (now no charge), and the glycine slows down. The chloride ions (negative charge) in the gel and sample buffers are flowing quickly towards the anode. This creates a high electric field and the SDS coated proteins (negative charge) are sandwiched between the fast chlorides and slow glycines, resulting in all proteins to reach the resolving gel at the same time. At the resolving gel (pH 8.8), glycine is deprotonated and negative again. Now the glycine run towards the anode faster than the proteins, and a low electric field is created. The proteins slow down, and flow through the pores of the gel based on their size, toward the positive anode. Small proteins flow faster, large proteins are slower. Here are some useful links that might help you understand pH and SDS-PAGE: - How does an SDS-PAGE gel really work? - SDS-PAGE (Wikipedia) Hope that helps! -Suraaj

How to Stain an SDS-PAGE gel

About

Search LabTricks

Members login panel