Gel Electrophoresis

Gel Electrophoresis

  • Why can't we run proteins on horizontal gel electrophoresis and DNA and plasmid samples on vertical gel electrophoresis?

    Why can't we run proteins on horizontal gel electrophoresis and DNA and plasmid samples on vertical gel electrophoresis?

    Ahmed ElFatih Amin Eldoliefy · North Dakota State University

    Both can run vertically and horizontally... check BioRad company Website...

  • Pablo Bolaños-Villegas added an answer:
    Which fluorescent DNA stain is the best for comet assay?
    Can anyone suggest a fluorescent DNA-binding dye that gives the highest signal to background ratio in comet assay. Also can anyone suggest a good software that is suitable for yeast comets' analysis.
    Pablo Bolaños-Villegas · University of Costa Rica

    SYBR Green

  • Deng Hongjing added an answer:
    What do you think about having more than one band than expected on a PCR gel?

    i have made a gel electrophoresis for a 7 samples after double arms pcr . the guide paper mentioned that the amplicon is 117 bp but i have seen more than one band in each well

    Deng Hongjing · Chinese Academy of Sciences

    Sorry, I can't see your picture. But I think other bands larher or smaller than the expect weight are non-specific band. It doesn't matter if you run RFLP markers. You can increase the temperature to make less non-specific bands.

  • sunil sharma added an answer:
    Running two SDS-PAGE in the same tank. One is running normally, the other is not. Any ideas?

    Both the gels have 5% stacking and 10% resolving. They are essentially copies of each other and they even have the samples. One of them is running while the other is not. I'm running them at 200V.

  • Omer F Celik added an answer:
    How do I interpret my urea-page results for proteolysis during?

    I would like to compare proteolysis rates, protein fractions and degradation products of beta and alpha caseins during the ripening period of cheese. I am wondering how you would interpret this data set? Please share your opinions and comments about these two urea-page electropherogram pertaining to two different types of cheeses.

    Omer F Celik · Ordu Üniversitesi

    Thanks Chanakya.

    Here are the densitometric values for 1st picture (we assume standards are 100 and measure the densities of other bands):

    BT      std 2nd 30th 60th 90th 120th
    Beta 100-122-75-65-57-56
    Alfa   100-16-11-12-10-10

    These are the densitometric measurements for 2nd picture:

    Control std 2nd 30th 60th 90th 120th
    Beta     100-116-83-63-53-46
    Alpha   100-48-20-22-21-16

  • Akhilesh Mishra added an answer:
    Which gel loading dye is suitable for DGGE?

    Hi, I am using promega master mix without dye so that equal concentration of PCR product can be loaded to DGGE. Now i did not understand which dye can be used before loading the sample. Can i used Gel Loading Dye, Blue (6X) from BioLabs? Please tell me the dye, what concentration and quantity should be used for loading?

    Akhilesh Mishra · University of Bedfordshire

    0.25% bromophenol blue

  • Nagarajan Dineshkumar added an answer:
    How can we make markers for 16S and 18S analysis on DGGE?

    Hi, can I make markers  for 16S and 18S analysis on DGGE? What will be the protocol? Can anybody suggest the easiest method?

    Nagarajan Dineshkumar · Independent Consultant, India

    Very few commercial DGGE reference ladders are available. http://nippongene.com/pages/products/electrophoresis/marker/dnamarker/dnamarker_e.htm (for the analysis of flora/fauna of bacteria, fungi and nematode in the soil)

    But making ladders on our own is the best choice. This article should help you. (Diversity of bacterial communities in container habitats of mosquitoes. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2904961/)

    There is no common reference ladder available for DGGE analysis. It varies with the environment or the sample you process. For my study, I have prepared and used a reference ladder of ammonia oxidizing bacteria from few type strains and environmental isolates of AOB.

  • Ganesh Shelke added an answer:
    Why am I not seeing banding on exosome western blot for Alix, Flot1, TSG101?

    I have attempted a western blot for SKOV3 exosomes and there are no bands present in the exosome sample (if there is they are in the incorrect MW location) and the cell lysate sample has nonspecific binding of the antibody. I used 18ug of cell lysate protein and 33ug exosome protein in each lane of the gel (NuPAGE 12%). For the sample preparation I used LDS sample buffer, RIPA buffer and protease inhibitor; vortex briefly, on ice for 10 min, vortex briefly then incubated at 70 deg C for 10 min then on ice until I loaded the gel. Transferred to PVDF membrane and visualized the protein separation with Ponceau staining. Then I used 5% NDMF to block for an hour and added the antibody to incubate overnight at 4 deg. The following day I rinsed with 1x TBST 3 times for 5 min each then the secondary ab (HRP) was incubated for 1 hr. The wash step was repeated and ECL substrate was incubated for 5 min then imaged using chemiluminescence. I am attaching a picture of the Ponceau staining (MW marker, CL, exosome; repeated 4 times total) and the resulting western blot with the antibodies. Any help would be greatly appreciated!

    Thanks!

    Ganesh Shelke · University of Gothenburg

    Hi,

    give a pbs wash to your extracellular vesicles (EVs) pellet to remove contributing serum protein (albumin) and do protein estimation again...by doing this u will get more  EVs protein per unit of total protein. Good luck.

  • Ren Jie added an answer:
    The band line of SDS PAGE is not straight. What is the problem, and how can I solve it?

    I ran 15 % of polyacrilamide for resolving gel, and the the wavy line comes out after 1 hour of running.

    Ren Jie · Xi'an Technological University

    If you use the  Ammonium persulfate change a new one 

    good luck

  • Mohammad mohsen Mohammadi added an answer:
    Can I get high purity DNA extraction from a 4% agarose gel?

    I want to separate two PCR products with 50bp apart (around 500bp). I’ve been trying to extract them using a 4% agarose gel and then the Thermo Scientific GeneJET Gel Extraction Kit. I am not having much success, is there a better kit to use for this agarose concentration, or other method that I should use instead?Thanks

    Mohammad mohsen Mohammadi · Shiraz University

    hi.do you try 1.5-2%. i think agarose 4% is not appropriate. try to use poly acrylamide gel.

  • Aleksandr Milshteyn added an answer:
    Does anyone have ideas for better resolution of 8kb-15kb DNA Fragments?

    I'm having trouble getting a good resolution in our restriction digests for bands in the 8kb to 15kb range. I would like to see +/- 1kb if possible. I am digesting a 45kb vector, so smaller fragments is not an option. 

    I have tried 0.5% agarose at 30V for 16 hours and was able to see separation, but the bands were not resolved enough (not sharp, and +/- 4kb).

    The next step I am considering is field inversion gel electrophoresis, but I need to buy a rig and this is outside my area of knowledge. I'm looking at the pippin-pulse.

    Any suggestions on how to get good separation in this size range? This is just for analysis, DNA does not need to be gel extracted. 

    Aleksandr Milshteyn · The Rockefeller University

    It comes with a few pre-set protocols that work pretty well.  The times that they demand are pretty high, but I've successfully cut the times down to ~50% by going down to 0.5% gel and increasing the voltage to the max 150 (~5V/cm in my gel box, their protocols are for 22.5cm interelectrode distance). I'm tempted to sit down and write a script for calculating size-range specific settings for it :)

  • Hovsep Ghazaryan added an answer:
    What does this RNA gel electrophoresis image indicate and where is mRNA?

    Hello All,

    This is the image of Total RNA non- denaturing gel electrophoresis. The 2 rRNA bands (28s and 18s) are prominent with 2:1 intensity but there is no smearing in between them that indicates presence of mRNA, so was the mRNA lost during extraction or what could be the possible reason for the disappearance of mRNA? Can I proceed the remaining Total RNA extract with In-vitro Transcription?

    Thankyou.

    Hovsep Ghazaryan · National Academy of Sciences of Armenia

    Dear Zainab,

    In this photo I see two fractions of rRNA, 28s on the top, 18s on the bottom. mRNA is not visible.

    Bests,

    Hovsep

  • Lori Roberts added an answer:
    Does anyone have experience with GelRed in Agarose electrophoresis? How does it compare to Ethidium Bromide?

    I am setting up my lab at a new institution and I am being encouraged to use GelRed for Agarose Electophoresis due to its safety. I have been told that, although the dye works, it usually alters the molecular weight of the bands and this is unacceptable for cloning experiments.

    I am wondering if anyone here has experience with this dye and what has been their experience.

    On other hand, many years ago I was taught that the Cancer risk associated with Ethidium Bromide comes from its property as an intercalating agent. GelRed is also an intercalating agent, therefore, it is logical to deduce that they might be equally risky. The manufacturers say that they are safe based on results of the Ames test. Is there any additional and independent evidence probing its safety?

    Lori Roberts · Biotium Inc.

    Hello, I’m a scientist at Biotium and would like to provide you with some information about GelRed that answers both of these commonly asked questions:

    1. "I have been told that, although the dye works, it usually alters the molecular weight of the bands and this is unacceptable for cloning experiments."

    2. "Many years ago I was taught that the Cancer risk associated with Ethidium Bromide comes from its property as an intercalating agent. GelRed is also an intercalating agent, therefore, it is logical to deduce that they might be equally risky"

    GelRed was designed with safety in mind, and therefore is a larger molecule than EtBr. This size means that is unable to cross healthy cell membranes and therefore unable to reach the DNA in living cells. It’s safety has been independently tested and verified and that information can be found here:

    http://biotium.com/wp-content/uploads/2013/07/GR-GG-Safety.pdf

    The fact that it is a larger dye also speaks to your first question. Because of its larger size, it can impact migration of DNA fragments when used as a pre-cast stain. Therefore, when very accurate information is needed about the size of a band, we recommend that researchers use the post-staining method, which can be performed successfully in as little as 10 minutes.

    As with EtBr, GelRed can be used for cloning and is removed from DNA during the purification process with a standard gel extraction kit.

    Here is some more food for thought: using a blue-light excitation source with a green dye like GelGreen gives as much as 100 to 1000-fold improvements in cloning efficiency compared to using a UV transilluminator by preventing UV damage to DNA during band excision from the gel.

    https://www.researchgate.net/publication/14283779_Protection_of_DNA_during_preparative_agarose_gel_electrophoresis_against_damage_induced_by_ultraviolet_light?ev=prf_pub

    I'll be happy to answer any other questions or concerns you may have about using GelRed or our other products!

  • Robert Shore added an answer:
    What is the minimum concentration of DNA needed, in order for bands to be observed on a 1% agarose gel after electrophoresis?
    I need to run an agarose gel that has restricted DNA however I need to know the minimum amount of dna that can be seen on the agarose gel. I presently do not get visible clear bands due to the concentration.
  • Aleks Po added an answer:
    What causes actin to "spread over" on Western blot?

    Hello my dear fellows,

    My Beta-actin for Western started to look a little strange. The bands look wide and kind of spread over, instead of being more narrow and compact. I am not sure what causes it.

    I was wondering if anybody has encountered this problem before and how did you go about fixing it

    Thanks!!

    Aleks Po · California State University, Channel Islands

    Hi Valeria,

    I decreased the voltage to 100V and it solved the problem.

    Thanks

  • Prem Subramaniam added an answer:
    Does it make sense to reuse buffers/chemicals in Molecular Biology?
    What is more efficient, to spare some chemicals and time for buffer preparation, or to make sure that the solution is always fresh? at which point is the buffer already too old? Any interesting examples of success/disaster with buffer reuse?
    Prem Subramaniam · Columbia University

    Hi Jan,

    Thanks for the reply!

    When one loses sight of the "first principles" of any method, you do not understand what you are changing and why.  A 'protocol" is set of fixed instructions that are linked together so that ALL steps work in an explainable fashion.  If you change one part, you better be sure that the other linked parts also work.  Just my 2 cents.......  :-)

  • David f Barker added an answer:
    How can I detect DNA on membrane to check the efficiency of Southern transfer?

    Hi all,

    How can I detect DNA on membrane to check the efficiency of Southern transfer? It is a pilot experiment that I am performing to set up things. I know I can incubate my gel in EtBr, but ideally I would like to see what's going on on my membrane as well. Is there a way to achieve that? Thanks a lot!

    Mina

    David f Barker · University of Louisville

    If the gel that you used for Southern blotting was stained with ethidium bromide, then the DNA that is transferred to the membrane will still be stained with ethidium bromide.  So if you take the membrane immediately after transfer (DO NOT perform any washing steps) and look at it over a UV light source, you should see the transferred DNA.  At the same time, you can look at the agarose gel that was transferred. If the DNA failed to transfer, it will still be present in the agarose gel.

  • Sobia Ejaz added an answer:
    What is the best way to prevent sds-page gel leakage?

    Hi all,

    I have tried many method such as parafilm, 5% agarose. My gel is still leaking. I have read the top comment on the youtube video (https://www.youtube.com/watch?v=b45nSOyPP_4). He suggested to line down the gel setting stand horizontally. Has anyone tried this method yet?

    But when I finish pouring resolving gel, I need to add water on the top to prevent oxygen get in. If I line it horizontally, I am not sure if the water would stay at the top part of the gel due to the gravity.

    Thank you

    Sobia Ejaz · H.E.J. Research Institute of Chemistry

    Make sure that the glass plates should not be broken or chipped from sides and leveled equally during the clamp. I always use rubber gasket below the gel plates. If you also use rubber gasket then make sure that it should be tight enough to prevent leakage. Thank you.

  • Rachel Ann Hauser-Davis added an answer:
    How to quantify each band in gel electrophoresis?
    I am doing DNA electrophoresis. I wonder is there a way to quantify the amount of DNA in each band
    Rachel Ann Hauser-Davis · Pontifícia Universidade Católica do Rio de Janeiro

    image J, photoshop, they're pretty much all the same, you count pixels and can then reach an approximate value for quantifyingyour bands in comparison to a known standard. I did BSA standards ranging from 5 to 30 ug (silver nitrate staining, if you stain by coomassie you should double or triple these values for 13 cm gels), and then added my samples to the other gel slots. When I scanned the gel, I just counted the pixels for each standard, plotted a standard curve of pixels x concentration and voilá!  Here: http://support.dalton.missouri.edu/index.php/wiki/Public:Quantifying_Color_Intensity/

    This is a very easy tutorial, you can easily do this on photoshop! 

    Cheers! :)

  • Marzieh Mahdavipour added an answer:
    Does anybody know why GelRed modifies the migration of DNA fragments in agarose gel electrophoresis?

    We have noticed that sometimes the migration in agarose gel electrophoresis  of DNA fragments is shifted to apparently higher molecular size when using GelRed to stain the DNA. This usually happens when analyzing PCR amplification products. It is not an intrinsic property of a particular DNA fragment because  in some reactions the mobilty is OK and in other samplkes is not. When the same samples are analyzed using ethidium bromide they have the same mobiltiy.

    Marzieh Mahdavipour · Avicenna Research Institute

    The other thing that I forget; I used Gel Red to post-run staining of the gel, but when I watch that with UV transluminator it had bright and white background, and the picture quality wasn’t good at all.

  • Adam B Shapiro added an answer:
    What can I do if the protein and DNA complex remain in the loading well in EMSA assay?

    The protein PI is 9.4, but the running buffer pH8.3, will this be the problem?

    Adam B Shapiro · AstraZeneca

    I think the smearing may happen because the protein and DNA are in equilibrium and dissociation of the complex occurs during electrophoresis.

  • Rakhee Rajput added an answer:
    Which buffer should I use for solubilization of hydrophobic proteins?
    I used a phosphate buffer but protein not dissolved. I attach 1D GEL scan. Bands are not resolved. There is lots of smearing.
    Rakhee Rajput · Indian Institute of Technology Delhi

    Thanks a lot all of you , finally I got my result.

  • Lea Guo added an answer:
    How can I cast separating and stacking gels without intermediate steps requiring overlay water?

    Hi All,

    I have read a protocol that say it is possible to cast separating and stacking gels without intermediate steps requiring overlay water. They mentioned adjusting the glycerol percentage in the separating gel. My separating gel solution is mixed between water+30%acrylamide mix+TrispH8.8+10%SDS+10%APS+Temed. It doesn't contain glycerol.

    I never do this and I'd like to know how.

    Thank you

    Cheerio,

    Lea Guo · University of California, Los Angeles

    Hi Asa,

    Instead of buying gradient gel, you can make two different concentration of separating gels on the same gel and add 10% glycerol to the separating gel stock for the bottom separating gel. You still need to add water/butanol upon pouring the upper separating gel, before stacking gel. Hope this help!

  • Maria De Lourdes Munoz added an answer:
    What are the reasons a PCR that once functioned, is not working anymore?

    Last week two Test-PCRs for a new set of primers worked quite well and i got one defined product band.

    One week later i am not able to get any product again. I exchanged all materials, also the template (cDNA) i also tried reamplification from pcr product of the test PCRs.

    Now i am wondering, why the pcr is not working anymore? Can Primers degrade within such a short time? Would it be senseful to run the primers on a gel electrophoresis?

    Thanks for your suggestions and your answers!

    Maria De Lourdes Munoz · Center for Research and Advanced Studies of the National Polytechnic Institute

    Your DNA probably is degraded.

  • Sachin A. More added an answer:
    How can I get the good bands on DGGE gel?

    I have isolated the soil DNA with power soil DNA kits. The soil was amended with and without biochar. I have done PCR, it shows nice bands on 1 % agarose, but when I run on DGGE, I missed bands in half samples. DNA concentrations were almost the same in all samples in the range of 13-22 ng/uL. The bands were visible in some samples with lower DNA quantity but not in higher. 

    Sachin A. More · University of Agricultural Sciences, Dharwad

    I would suggest you to load different concentration of pcr of product (lower to higher) of only one sample (in which your getting good amplification) on dgge gel and follow the silver staining method to visualize the gel bands as it is very sensitive method ranges from pg to ng. once you standardize the concentration of pcr product required to load on gel then use the same concentration of pcr product  of all samples to load on gel. Before proceeding towards dgge gel always confirm that sample is getting amplified correctly followed by gel quantification of pcr product and then accordingly load the volume each pcr product on dgge gel. Some times bands will not be separated properly on dgge gel. I n that case try different  gradient of denaturing % and as well as page gel % (acrylamide : bis acrylamide). good luck.... 

  • Md Moniruzzaman added an answer:
    What is the value of the dielectric constant/relative permittivity of electrophoresis gel?
    I am trying to simulate (in Comsol) electric field through an electrophoresis system and need an approx. value for the dielectric constant/relative permittivity of any kind of gel (like PAGE or agarose) etc. Any ideas?
    Md Moniruzzaman · Dongguk University

    For SDS-PAGE it is 100 V for 90 min. You can change the volt from 90-110 and also the time point. but the time should not be less than 90 min.

  • Mason Sweat added an answer:
    Why do I see my protein ladder standard on my developed western blot?

    I'm attempting to use a western blot to detect the presence of an endogenous transcription factor. After I have shown that my western conditions are capable of detecting the transcription factor, I will use co-ip to show that the transcription factor is complexed with a separate protein.

    Obviously, I need to be able to detect the protein in a western first!

    My conditions:

    1. Scrape cells from a 60 mm dish in 1 ml of pbs, pellet, aspirate pbs.

    2. Lyse cells in Tropix lysis solution (ABX210LM), and add 1) general protease inhibitor (sigma) and PMSF (5 ul of each)

    3. Add SDS loading buffer, boil 5 mins, load SDS-PAGE gell

    4. Semi-dry transfer the gel onto the membrane (not nitrocellulose but the other kind, drawing a blank on that sorry). Ensure that my ladder standard is visible on the membrane.

    Blocking: 10% carnation milk 1 hr room temp, slow rock

    5. Primary antibody 4 degrees celcius ovn (1:1000, pbst)

    3x wash pbst

    6. Secondary antibody 4 degrees 1/2 hour (1:15000, pbst)

    3x wash

    7. ECL prime, 5 min rt, exposed

    8. blot dry and developed.

    Any thoughts are appreciated!

    Mason Sweat · University of Iowa

    Thanks to all!

  • Manchala Nageswar Reddy added an answer:
    Why do loading dyes run quicker than the protein loading sample?

    Hi all,

    I tried using 15% gel SDS for running vertical SDS-PAGE gel electrophoresis at conditions: 100V, then 120V after loading sample is lining.

    I found out that the loading sample/dye (mixing with 5x loading dye in ratio 1(dye):5(total) and boiled at 95°C for 5 min) ran down much faster than the prestained protein marker does; the dye is near at bottom of gel while prestained protein marker is still at half of the gel only. When stained with Ponceau S, loading protein running down only half of the membrane.

    I usually use 10% gel and stop at the dye front are a few cm above bottom of the gel. The dye and the protein are running about the same speed.

    My question is whether loading dye's running down quicker than protein loading sample. Don't they suppose to run down together? What are the causes?

    Thank you for your answers

    Manchala Nageswar Reddy · Central University of Kerala

    check the components of loading dye.If possible prepare a fresh dye and run the samples.

  • Nicolas Viphakone added an answer:
    Alternatives to Trans-Blot Turbo PVDF packs
    My lab has a Trans Blot Turbo from Bio-Rad and as far as I can tell you can only get the high speed transfers by ordering their proprietary blotting paper/buffer? I feel bad spending so much on the preassemble packages. Has anyone ever tried to get the rapid transfers on their own?
  • Jürgen Fritsch added an answer:
    Can anyone help me in protein ubiquitination detection?

    I'm currently working on a protein which is ubiquitinated for sure. Next step is to identify if it undergoes K-48 or/and K63 ubiquitination. I over-expressed my protein in combination with HA tagged Ub-K48 only or Ub-K63 only expressing plasmid, pull-down the target protein under denaturing conditions and then probe the sample with anti-HA antibody in immunoblotting. I always found a weak band corresponding to Ub(1) by using Ub-K48, and just one or two bands when Ub-K63 is present. I was wondering if anyone can help me to interpret these results. Dose anyone have an alternative protocol to assess ubiquitin moiety characterization?

About Gel Electrophoresis

Gel electrophoresis is a method for separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments, based on their size and charge.

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