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Expected bands in polyacrylamide gel


I have something in my notes that doesn't seem right.

If I use restriction enzymes on a PCR product how many bands should I see provided I know the restriction enzimes will cut the DNA? If I use two restriction enzymes I expect two cuts then then gel should show three bands. Then, if I use just one enzyme and I know the enzyme will cut just once, then should I expect two bands? According to my notes one band is expected.


If your restriction enzyme only targets a single site on the DNA sequence then it will split each DNA strand into two fragments. If the restriction enzyme cuts every strand then you will see those two fragments. Note that if the two fragments have the same length they could appear as a single band when separated using gel electrophoresis.

If the enzyme doesn't cut everything the uncut DNA will also be present. This is why you should always separate some uncut DNA in the gel electrophoresis.


Technologies to Engineer Cell Substrate Mechanics in Hydrogels

Makoto Funaki , Paul A. Janmey , in Biology and Engineering of Stem Cell Niches , 2017

8.1 Polyacrylamide Gels

Polyacrylamide gels have served as an important tool to investigate the effect of substrate stiffness on cellular functions in various cell types since Pelham et al. reported that cell motility and focal adhesion in fibroblasts are regulated by the stiffness of collagen-coated polyacrylamide gels. 62 One of the advantages of polyacrylamide gels is that they are biologically inert. As a result, tuning the stiffness of polyacrylamide gels by adjusting the concentrations of acrylamide and bisacrylamide, which affects the density of the polyacrylamide network, does not influence the biochemical property of the gels. Thus, it is possible to assume that any difference in cellular functions observed between cells seeded on polyacrylamide gels with different stiffness is attributable to the difference in the stiffness of gels. By varying the concentrations of acrylamide and bisacrylamide, the range of stiffness can cover that of most soft tissues ( Fig. 23.2 ). 63 However, this biological inertness of polyacrylamide prevents binding of cell surface receptors and adhesion molecules present in the medium. Thus, to engage in cells, adhesive molecules need to be covalently linked to the gel by using cross-linkers, which have two functional groups one binds to polyacrylamide and another binds to adherence molecules, such as collagen and fibronectin. 6,64 One disadvantage of polyacrylamide gels is its limitation to 2-D culture because acrylamide is highly toxic before polymerization.

Figure 23.2 . Mechanical properties of polyacrylamide substrates.

The shear modulus of polyacrylamide gels with a range of acrylamide (indicated as percents near data lines) to bis-acrylamide (indicated as cross-linker) proportions was measured. The shear modulus (G), expressed in Pascal, increases at constant polymer mass with increasing cross-linker. Increasing the concentration of acrylamide from 3% to 12% also creates a large stiffness range from 10 to 50,000 Pa. The solid line denotes the theoretical stiffness of a rubberlike network if every cross-link was elastically effective.


PAGE (Polyacrylamide Gel Electrophoresis), is an analytical method used to separate components of a protein mixture based on their size. The technique is based upon the principle that a charged molecule will migrate in an electric field towards an electrode with opposite sign.The general electrophoresis techniques cannot be used to determine the molecular weight of biological molecules because the mobility of a substance in the gel depends on both charge and size. To overcome this, the biological samples needs to be treated so that they acquire uniform charge, then the electrophoretic mobility depends primarily on size. For this different protein molecules with different shapes and sizes, needs to be denatured(done with the aid of SDS) so that the proteins lost their secondary, tertiary or quaternary structure .The proteins being covered by SDS are negatively charged and when loaded onto a gel and placed in an electric field, it will migrate towards the anode (positively charged electrode) are separated by a molecular sieving effect based on size. After the visualization by a staining (protein-specific) technique, the size of a protein can be calculated by comparing its migration distance with that of a known molecular weight ladder(marker).


DNA staining in agarose and polyacrylamide gels by methyl green

Methyl green (MG) is an inexpensive, nonproprietary, traditional histological stain for cell nuclei. When bound to DNA and upon excitation with orange-red light, it fluoresces brightly in the far red region. We compared MG with ethidium bromide (EtBr), the conventional stain for DNA in gels, and Serva DNA stain G™ (SDsG), a proprietary stain marketed as a safer alternative to EtBr for staining of electrophoresed DNA bands in agarose and polyacrylamide gels. DNA-MG fluorescence was recorded and 2.4 μg/ml MG produced crisp images of electrophoresed DNA after incubation for 10 min. Stain solutions were stable and detection limits for faint bands as well as relative densitometric quantitation were equivalent to EtBr. MG, EtBr and SDsG cost 0.0192, 0.024 and 157.5 US cents/test, respectively. MG is an effective stain for visualizing DNA in agarose and polyacrylamide gels. Its major advantages including low cost, comparable quality of staining, storage at room temperature, photo-resistance and low mutagenic profile outweigh its disadvantages such as staining of tracking dye and requirement for a gel documentation system with a red filter.

Keywords: DNA binding Serva DNA stain G™ ethidium bromide far red fluorescence gel documentation system intercalating agent methyl green molecular biology.


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Bassam, B.J. & Bentley, S. Electrophoresis of polyester-backed polyacrylamide gels. Biotechniques 19, 568–573 (1995).

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Gel electrophoresis method to measure the concentration of single-walled carbon nanotubes extracted from biological tissue

A rapid and sensitive method to detect single-walled carbon nanotubes (SWNTs) in biological samples is presented. The method uses polyacrylamide gel electrophoresis (PAGE) followed by quantification of SWNT bands. SWNTs dispersed in bovine serum albumin (BSA) were used to develop the method. When BSA-SWNT dispersions were subjected to sodium dodecyl sulfate (SDS)-PAGE, BSA passed through the stacking gel, entered the resolving gel, and migrated toward the anode as expected. The SWNTs, however, accumulated in a sharp band at the interface between the loading well and the stacking gel. The intensities from digitized images of these bands were proportional to the amount of SWNTs loaded onto the gel with a detection limit of 5 ng of SWNTs. To test the method, normal rat kidney (NRK) cells in culture were allowed to take up SWNTs upon exposure to medium containing various concentrations of BSA-SWNTs for different times and temperatures. The SDS-PAGE analyses of cell lysate samples suggest that BSA-SWNTs enter NRK cells by fluid-phase endocytosis at a rate of 30 fg/day/cell upon exposure to medium containing 98 microg/mL SWNTs.


"Wavy" bands in SDS-PAGE gel - (Jul/19/2013 )

I've been running SDS-PAGE gels this summer for the first time trying to separate myosin heavy chains present in skeletal muscle. I've noticed that sometimes I get uneven and wavy bands. Sometimes this is actual curvy-ness in the band, sometimes it's just the band tilting in the lane. I can get gels where I have no waviness at all, but I haven't figured out what I'm doing differently. Is the waviness just due to tearing the wells? Or possible bubbles at the bottom of the separating/resolving gel?

For my gels, I run two gels at the same time on an Hoefer SE600 system. Because I'm separating such heavy proteins, I run the gel at around 7C for about 45 hrs. My voltage in the stacking gel is 70V and then I increase it to 200V in the separating/resolving gel.

Here are two gels I ran yesterday, both illustrating my issue:
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if it is a crude extract it may contain salt which disturb performance of SDS-PAGE

But it doesn't always happen with the same samples.

For example, in the first gel image, lanes 3, 7, and 13 are all exactly the same thing, but only lane 7 has a wave.

You can also check out this SDS-PAGE &ldquoHall of Shameful Gels&rdquo http://www.ruf.rice.edu/

Thanks for the tip. I had seen the hall of shameful gels before, but I'm fairly certain my stacking/separating interface is clean and straight. I also rinse out my wells with running buffer a few times before loading.

I'm going to try letting the gels polymerize for a bit longer, as well as ensuring wells are straight and I don't remove the comb to quickly or roughly. We'll see if that helps.

in addition to the advice in the linked post, if you are going to run the gel at 7C then you will have to use lithium dodecyl sulfate (lds) instead of sds. sds crystallizes at lower temperatures and can cause all sorts of mischief.

we used to separate myosin heavy chains (from brain) by 3.5-5% gradient gels with an 8-0M urea gradient overnight runs at 5mA (i think, it was a very long time ago).


Expected bands in polyacrylamide gel - Biology

Native PAGE Principle:

Native PAGE uses the same discontinuous chloride and glycine ion fronts as SDS-PAGE to form moving boundaries that stack and then separate polypeptides by charge to mass ratio. Proteins are prepared in a non-reducing non-denaturing sample buffer, which maintains the proteins' secondary structure and native charge density. Therefore you can easily see multiple bands from the camshot of your native PAGE gel if your target protein has polymerized forms in your sample. In native PAGE electrophoresis most proteins have an acidic or slightly basic pl (isoelectric point) (

3–8) and migrate towards the negative polar. If your protein's pl is larger than 8,9, for example, you should probably reverse the anode and run the native PAGE gel.

Learn more about Native-PAGE:

  • For the electrophoresis system, a bio-rad system is recommended.
  • For a 5ml native PAGE stacking gel

*: Added right before each use.

For a 10ml native PAGE separating gel:

Acylamide percentage 6% 8% 10% 12% 15%
Acrylamide/Bis-acrylamide (30%/0.8% w/v) 2ml 2.6ml 3.4ml 4ml 5ml
0.375M Tris-HCl(pH=8.8) 7.89ml 7.29ml 6.49ml 5.89ml 4.89ml
*10% (w/v) ammonium persulfate (AP) 100μl 100μl 100μl 100μl 100μl
*TEMED 10μl 10μl 10μl 10μl 10μl

*: Added right before each use.

Sample buffer (2x):

62.5 mM Tris-HCl, pH 6.8
25% glycerol Glycerol
1% Bromophenol Blue

25 mM Tris
192 mM glycine

Note: running buffer should be

pH 8.3. Do not adjust the pH.

Gel running protocol:

1. Prepare appropriate amount of separating gel in a small beaker, then add specific vol. of AP and TEMED and gently swirl the beacker to ensure a sufficient mixing. Pipet the gel solution into the gap between the glass plates of gel casting (Don't fully fill). Fill the rest space with water (isopropanol alternatively). Allow 20-30min for a complete gelation.

2. You can prepare the stacking gel solution while the separating gel is gelating. Prepare appropriate amount of stacking gel in a beacker and mix with 10% AP and 1% TEMED. Pour out the water in the first step and pipet the stacking gel solution into the gap and insert the comb. Allow 20-30min to let it gelate.

3. Mix your sample with sample buffer. Do not heat your sample!

4. Load the sample mixture and set an appropriate voltage to run the electrophoresis.

Note: It's better to put the system on ice and not set a relative high Volt in case the proteins degrade.

5. Stain as you would a standard Coomassie-blue protocol or proceed to a immuno-blotting procedure (western-blot).


Note : Before running the gel make sure that the gel, gel apparatus and samples are ready.

  1. To assemble, take out the gels from the casting frame and clamp them in the gel apparatus.( Make sure that the short plate always faces inside and if you have got only one gel to run use the dummy plate that is available to balance).
  2. When the plates are secured, place them in the cassette and then lock it.
  3. Place them in the gel running tank.
  4. Fill the inner chamber of the tank with buffer.(Now it is easy to remove the comb, since it is lubricated).
  5. Remove the comb CAREFULLY(without breaking the well).
    [Now the gel is ready to load the samples]
  6. Rinse the loading tip a few times with distilled water. (Make sure that all the water is poured out before loading the samples.)
  7. Insert the loading tip to a few mm from the well bottom and deliver the samples into the well. Rinse the syringe with distilled water after loading for a few times .
  8. Attach the power supply by putting the lid (Make sure that the connection is in correct way ie., black - black and red - red). Set the voltage upto 180 V and run for 1 hour.(Don't allow the dye front to go out of the gel).

CONCLUSIONS

The use of dyes and other alternative materials in simulating DNA gel electrophoresis provides many benefits beyond cost savings. Most materials described may already be available in most school laboratories or should be readily purchased through general laboratory suppliers. Time and effort can also be saved by omitting the need for preparing actual DNA samples. Even more valuable classroom time is saved by eliminating the additional steps needed to stain and destain DNA gels to visualize the bands while enhancing laboratory safety.

The use of dyes also transforms gel electrophoresis into a “real-time,” visually intuitive demonstration of the process and outcome of electrophoresis. Since the dyes visibly migrate and separate within the first few minutes, students can be engaged quickly and find it easier to maintain interest whilst the instructor encourages exploration during the time taken for electrophoresis with appropriate questions. There is no need to wait till after staining the gel to view the effect of electrophoresis.

The benefits of using dye-based gel electrophoresis appear to be widely appreciated, going by various informal accounts and anecdotal testimony by school students, teachers, and undergraduates conveyed to the authors. We present here a complete set of effective substitute materials for simulating DNA gel electrophoresis in schools. It is also hoped that it will support educators in devising their own useful and exciting hands-on teaching protocols.


Watch the video: 6CCM111-Electrophoresis2021 (December 2021).