Science method
Polyacrylamide Gel Electrophoresis - Science method
Polyacrylamide gel electrophoresis is a method for separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments, based on their size and charge, using polyacrylamide as a gel medium.
Questions related to Polyacrylamide Gel Electrophoresis
I am running a DNA PAGE after PCR (samples 6-15 are run in duplicate with the second sample digested) to determine serotonin genotypes. The ladder (well 1) is on the far right of the attached image). I would greatly appreciate any advice on how to enhance band brightness and definition, thanks.
Additional information: 5 uL ladder added, 10 uL PCR product per well, PH of the buffer is correct. Temperature of the room ~75F with Gel container NOT on ice.
Good evening, I was wondering what is the size of Acrylamide/Bis-Acrylamide in a PAGE 4%/8%. I'm asking because after the run I found that some of the sample stayed in the well
Can we add midori green directly while preparing polyacrylamide gels? or should only be done as a post staining after running the PAGE gel?
I’m currently working on the synthesis of aptamers through SELEX and the technique I’m using for converting dsDNA to ssDNA is asymmetric PCR, however when performing PAGE Denaturing electrophoresis, I cannot see any product bands, just the primer, why is that? for the asymmetric PCR I use only FAM reverse primer 1uM.
I'm attempting to separate two DNA oligos of length 120bp and 100bp. I've been running them with 2x formamide loading buffer for 3 hours at 65V. I understand constant power is important for the temperature of the running buffer to help denaturation. My lab only has a constant Volts or Amperes power supply, so this is what I've been using. I've been considering purchasing a more advanced power supply that has constant power. How important is this for overall denaturing PAGE settings? Is there anything wrong with my current settings? My current method semi-works and I'm considering continued troubleshooting or exploring constant power.
If yes, then how can I interpret the results?
If yes, then how can I interpret the results?
I've been doing miRNA northern bloting for over months. I have a question: Dose anybody know whether loading of total RNA with EB and run in denatured PAGE gel (15%, urea) cause shifting of the microRNA bands or not? I stained the gel post electrophoresis but the bands are poorly visualized; therefore I load total RNA with EB then run the gel, I found the signals of the rRNA and tRNA bands are very strong, but after hybridization of the transferred membrane, I found the band signals of the target miRNA as well as the artificial miRNA appeared in inappropriate position. They should be appeared in between the xylene cyanol and bromophenol blue bands. Could anybody tell me if I make it wrong? Why?
I am studying DNA-ligand interactions with EMSA. Sometimes I cannot see the DNA bands in the PAGE gel when I stain it with Etbr. As silver staining is more sensitive to EtBr, I have to use the silver staining method. This can be time consuming. Is it possible to stain DNA with EtBr first and then stain the same sample with silver?
Hello,
I don't understand the difference in what you can see after running your DNA on an urea or an alkaline gel. I know how alkaline works: high pH, hydrogen bonds are prevented, as a result your DNA migrates as ssDNA. But as far as I understand, it is the same with urea: you denature secondary structures with urea, but in the end it is also used to purify or analyze ssDNA.
I already run multiple urea PAGEs and I could distinguish between different ssDNA molecules on the gel. Now my supervisor told me to run an alkaline gel. However, I do not understand what additional information it would give me, since both are denaturing gels? How do you decide to run an urea or an alkaline gel?
Why is there background on my gel. It is due to Coomassie that will not destain. How do I eliminate it, and what is causing it?
I am using a PCR amplified product of around 56 bp with a T7 Promoter sequence and I am getting a single band after PCR. However, after in-vitro transcription of this PCR Product, I am getting two distinct bands on 15% Urea PAGE gel. I tried optimizing the reaction using Mg2+ and rNTP gradient. What should I do?
What could be the possible reason for not getting proper band of ladder while performing PAGE
I am trying to visualize labeled oligonucleotides with UREA PAGE, but I do not see any band. Do I have to change the wavelength of excitation ?
I'm trying to attain kinetic parameters of TdT. And I'm using Fam-labeled oligo. I made 20% TBE-UREA PAGE, with 8 M urea. I pre-run my gel and load only 5 microliter of sample. The first four lanes from left are my oligonucleotide with incubation with enzyme and the last lane is my control without enzyme. I want to get discrete bands , what should I do?
Hello everyone,
I wanted to visualize my DNA fragments after PCR in Non-denaturing PAGE, however I could not. Because amper was not increased during the PAGE assay.
what are the causes of not increasing amper in Non-denaturing PAGE?
(I used freshly prepeared 5X TBE (PH:8.29). I used Bıo-Rad Mini-PROTEAN® Tetra system for PAGE asssay)
Thank you in advance.
We're using a 15% PAGE gel to separate a 98 vs 118 bp oligo, but are struggling to obtain adequate yields using crush and soak and various diffusion-based kits. We are considering trying electroelution, and one of the products we've found are G-capsules. Most papers using them seem to use them to elute genomic DNA from low-% agarose gels. Has anyone here tried them for something similar to our application?
After starting the electrophoresis my samples often 'fall' trough the pocket of the gel and accumulate on the stacking/resolving gel border. Every ingredient of the gel had been prepared fresh, the gel was allowed to polymerize long enough and was also stored correctly. Had anyone similar problems and knows how to solve this?
Thank you for your advice.
I know that both Orange G and Bromophenol blue are negatively charged dyes and that Orange G may migrate faster in an agarose gel.
I found a resource that says Orange G can be used in Native-PAGE and in a DNA PAGE but I cannot find any resources that say whether the two dyes are interchangeable in a protein SDS-PAGE.
Any insight would be greatly appreciated.
Hello all,
I have been trying to extract RNA from polyacrylamide gel and I am consistently getting low A260/230 ratios (0.7-1.2) and this interferes with my downstream application. I believe this impurity is linear polyacrylamide. I have tried a few different procedures and also tried using the Zymo ZR-small RNA PAGE extraction kit. Has anyone ever faced similar issues? I would appreciate any suggestions. Thanks!
When I casting Urea PAGE gel, I can see the gel shrink in bottom and no wells formed but gel solidified. Does anyone know a possible reason or how to minimize this?
My protein of interest is a bacterial inner membrane associated protein. And I suspect it to be forming an insoluble aggregate in a mutant strain. I read that Blue Native PAGE works better than just Native PAGE for membrane proteins. Is that true? I was planning on running a Blue-Native PAGE and then doing a Western with it to test if my protein forms an aggregate. Alternatively, is it better to just run a Native PAGE here instead of Blue Native PAGE?
I appreciate in advance all your help/guidance/comments addressing my question.
PS: We have a BIO-RAD mini gel apparatus in the lab, so I would love to have a protocol that works with this system.
I just ran a and SDS PAGE with 12% resolving gel and 4% stacking gel. The problem is that as soon as I turned on the electricity (180V), the samples moved down the stacking gel in a wiggly manner. Then when they reached the line between the stacking and resolving gels, the samples dispersed. I continued running the gel but after the staining I did not get any bands except for my standard ladder. Has anyone experience such a thing before?
I am carrying out polymorphism studies using ssr markers and running them on 6% polyacrylamide gel using 1x tbe buffer. But I'm facing smearing type of bands and multiple bands in certain samples can anyone kindly answer why this problem is encountering
I run my gene specific primer PCR product on 2.5% Agarose it did not show any amplification only primer dimer was seen
But the same product I used in 8% PAGE gel (with silver staining process) it shown the amplification result
What is the reason behind it...
How can I separate two DNA fragments of 3000bp and 3700bp using polyacrylamide gel electrophoresis?
I prepared a large number of protein extract samples for polyacrylamide gel electrophoresis with bromophenol blue to track migration through the gel. I have also tried (unsuccessfully) to detect a protein of interest in these samples by ELISA. I suspect the bromophenol blue is interfering the protein's interaction with the high binding plate. Has anyone experienced this problem before?
Dear all,
I am trying to do an EMSA experiment with ssDNA. I am redoing an EMSA that was published.
The gel is a small (10 x 8 cm) 10% polyacrylamide (37.5:1), TAE (40 mM Tris pH7.6, 2.5 mM EDTA)
The binding buffer is 10mMTris pH7.6, 50 mM KCl, 1 mM DTT, 10 ug/ml BSA and 20 nM ssDNA oligos. The binding reaction is done at 20C for 30 min.
The gel is pre-runned at 45V for 1hr. in running buffer (10 mM Tris, 50mM KCL and 2 mM EDTA)
after binding reaction 10 ul was loaded on gel and run for 2 hrs at 45V.
What is going on with my gels? is the gel getting too hot? I do run the gels in the cold room or in an iced box. Please someone give me some pointers? Thank you!
Johannes H Matse
I am doing in vitro transcription, my target RNA is 36nt in length. After transcription when I am running it on Denaturing gel, I am not able to see my RNA band under UV shadowing. But when I am staining the PAGE gel with SYBR gold I am observing the RNA band at correct length. For Cutting the RNA band and it's purification, I need to cut the band under UV shadowing. I have attached here the gels with and without staining.Any suggestions will be highly appreciated.
I have run mRNA on 1.5% TAE gel but the band is ~4kb above its actual size. I have heat my sample at 90oC for 2 mins to avoid secondary structures. But I haven't seen the mRNA on actual size.
I have been seeing this weird looking gels. I am running RNA in 15% PAGE for 90 min at 90 V. Have anyone of you come across this situation? Can someone suggest me any idea to resolve this?
My 15% PAGE recipe is Acrylamide = 3.75 ml, TBE Buffer (10x) = 1 ml, 10% APS = 0.25 ml, TEMED = 15 ul, Water = 5 ml.
Hello everyone
I am looking for a PAGE gel protocol/recipe for RNA.
Hello everyone,
I am relatively new to Western blotting and am having problems with transfer efficiency (wet). Some protein has been getting transferred from the gel to the membrane based on my Ponceau S staining but not very much. When we visualize with fluorescent antibodies, I have only been able to see fluorescence for actin and not any of our other proteins which should be expressed relatively higher in our mutant strain.
Recently, I ran 2 gels and stained 1 with Coomassie blue before transfer and 1 after transfer. The protein bands seem about the same intensity/size on both except for the protein ladder. The lane with the protein ladder left no bands after transfer expect for 1 around 17kDA.
I have been using Towbin's transfer buffer (192 mM glycine, 25 mM tris, 20% methanol) and the Mini-PROTEAN Tetra Cell for transfer. The membrane type is 0.45 uM PVDF membrane which I have been soaking in methanol for 10 min and then TTB for 5 min. All other transfer cassette sandwich materials, including the gel, I equilibrate in TTB for 15 min.
I have tried transferring overnight at 4C and 25V and for 1 hr at RT and 100 V with similar results. During the last transfer, I also recorded the starting and ending current (start-231 mA --> end-367 mA). 1 hr RT w/ ice pack and stir bar. The TTB I used I made that day and chilled in the fridge for about an hour.
Are there any red flags in my protocol that might be affecting my transfer?
Any help would be appreciated, thank you!
Hi all,
I am trying to purify from PAGE gels (with or without UREA) RNA/DNA fragments which lengths are between (for instance) 40 nt and 60 nt.
What I though was to produce some sort of home-made ladder that shows 2 bands for the limits I need and then cut between these two (in another well of course). Therefore I ordered 2 DNA primers, one of 40 and the other of 60 nt and I run them on a PAGe gel to see how they are migrating. What I got was only a smir (see figure).
Is this because my primer was perhaps too concentrated or I am just doing something silly? Do you have better suggestions on how to track specific lengths on a PAGe gel?
Thanks!
I want to do a toeprinting assay to map translation start sites. Typically, these experiments are carried out with radioactively labeled oligos that are extended using reverse transcriptase. However, working with radioactivity is difficult in my institution, and I would like to avoid it.
I am thinking of an alternative. Specifically, what I am planning to do is to use 5'-biotinylated primers, then employ reverse transcriptase as in the standard protocol, then run the primer extension products on a PAGE gel, then transfer it onto nitrocellulose membrane just as if it were a Western blot (I would use the exact same protocol, just leaving out the SDS), and finally stain the membrane with streptavidin-HRP.
To my surprise, I nowhere found a protocol like this in the literature. Instead everybody keeps working with radioactively labeled primers. This suggests to me that my plan is probably a bad idea, because somebody must have tried this, right?
If anyone would like to way in, I would appreciate an opinion. I'd be happy about any support for my experimental layout, any concerns why this might/will not work, or suggestions on how I could optimize the plan ...
I am doing DNA PAGE. Buffer is TBE. I controlled the buffer in agarose gel electrophoresis system (in gel and also as buffer) but it is working. I can see sometimes bubbles but the voltage needs to be max. 300 V. What should I do?
Thank you
Hi...can anyone suggest me....
Related to PAGE. (Polyacrylamide Gel Electrophoresis) and it's protocol..How much voltage and current need to keep during PAGE run and why?..... Why actually PAGE is preferred much over normal agarose gel for polymorphism? ..Dry run need to be done for how long? . . .
Role of APS ,,TEMED and ABS in PAGE...
We are using NuPAGE gels to resolve protein samples, and the included protocol calls for the addition of 0.5mL "antioxidant" to the inner gel chamber. I understand the purpose of the reagent, but is anyone aware of a recipe to prepare an appropriate substitute?
I tried to synthesize RNA from a linear DNA template, but I found there were always RNAs of various lengths that got synthesized after repeating the experiment two times. I have added RNase inhibitor every time but still got many RNA bands. And I run the RNA in denaturing PAGE. Does anyone have any suggestion or explanation of this problem?
I am having a recurring issue with the incomplete transfer of protein bands from my PAGE gel to the PVDF membrane. SOME proteins transfer, but many do not. These bands that aren't transferring are prominent bands, so there has to be a significant amount of protein there. I have attached a picture (the stain-free PAGE gel is in the left and the blot is on the right. The red arrows indicate just a couple of significant bands that are missing.).
The transfer protocol is 16 hours at 35V constant, 90 amps. Transfer buffer is Towbin without SDS.
Any suggestions would be appreciated!
I'm trying to replace my small RNA prestain ladder with a much cheaper tracking dye that I can make up myself. Has anyone any suggestions. I run 10% urea-PAGE gels for 75 mins at 300V. I tested Bromophenol blue and Xylene Cyanol recently and Bromophenol blue co-migrates with RNA fragments that are too small so it runs off the gel before it is useful. The Xylene cyanol co-migrated with 30bp RNA which is great. I'm looking for something that will also co-migrate at the 20bp mark too as we use this mark as a guide when to stop the gel. I was wondering if cresol red is useful but haven't seen it used on a 10% PAGE gel. Thanks in advance.
Hi RG community,
I'm about to order some single guide RNAs (gRNAs) for CRISPR/Cas9 site-directed knockouts following Bassett and Liu (2014) protocol (paper below). Briefly, two partially overlapping primers (an ~ 64 nt forward target-specific and an 80 nt universal reverse) are used in PCR amplification to generate a DNA template, which will then be used for T7 in vitro transcription (gRNA synthesis).
Now, I've always ordered these primers PAGE-purified, reasoning that more full-length oligos (non-truncated) would generate more full-length functional gRNAs, thus getting more mutagenesis efficiency. However, some colleagues have been using standard de-salted oligos instead, and they have the impression that works just fine. Considering that they're way cheaper than PAGE-purified oligos, it might worth a try, but I'm very skeptical about it.
So, that's the question: are PAGE purified oligos really necessary for CRISPR/Cas9 mutagenesis?
Any and all comments are very appreciated!
Dani.
Bassett and Liu (2014):
I’m looking for a simplified protocol for a Blue-native PAGE. Can someone please share their thoughts and experience. Thank you.
hi
i am running a native page to measure the amount of bovine serum IgG.
i follow the protocol (http://www.ispybio.com/search/protocols/www_assay_protocol_com_molecular_biology_electrophoresis_nat.pdf )
whether i run PAGE for one hour or two hour, protein is placed near to loading channel like the photo.
does pH affect the protein, so that it has no charge..?
or is acrylamide-bis percentage so high that pore size is small to pass
thank you for reading.
Does pre-running the PAGE gels useful/helpful? Does it affect the resolution of bands? If yes, what might be the reason. Asking this because I've got both positive and negative reviews on this.
I intend to dilute my protein samples in PBS but am concerned since this is a different buffer system compared to the Tris buffers used in PAGE. I would like to ask if suspending my proteins in PBS, aliquoting it, and mixing with electrophoresis sample buffer is just fine.
Dear collegues,
The final aim is common: to band cut distinct PCR products from my gel slice after PAG vertical electrophoresis folowed by purification/elution. However, I could not obtain specific well defined bands in my gel in spite of well known capability of this approach to divide PCR amplicons few bp distinguished. I attached the picture of my gel (see the 3rd well). What am I wrong pursuing the ideal picture with defined sharp bands, whether it is feasible at all?
Gel composition: stock solution (20% acrylamide, 5% bis-acrylamide) diluted in 3.3 times, so the final concentration is 6%. Both Persulfate ammonium and TMED are used for crosslinkage.
Device parameters: Bio-rad chamber and power supply on impulsed protocol: 50V for 30' followed by 150V for 60'.
Product size: 870 bp
Hi everyone,
I am interested in performing fingerpitng DNA tests with ISSR and RAPD markers. Is a special purification of the primers needed (PAGE or HPLC) ? Can the type of purification affect the result? can I use the purification that is perform by default (MOPC)?.
Thank you in advance.
I'm working with very short sequences of ssDNA (~20 nt), and, when I run PAGE gels (Novex TBE, 20%), I frequently observe bands that look like bubbles. The attached image shows what happens when we run for about 2.5 hours at 80 V (we pre-run for half an hour). We've also tried larger voltages with no avail. I know this is somewhat of a niche use case for these gels, but does anyone have ideas on what's going on/how to prevent this? Thanks!
I'd like to perform single-strand conformation polymorphism (SSCP) in my thesis, however I cannot control the temperature of the vertical PAGE since we are using the conventional tanks. Is there a way that I could somehow lower and monitor its temperature?
Hi, I'm trying to produce ssDNA from PCR. I want to use it for SELEX procedure.
The idea is to use polyA tail tagged reverse primer. I hope I can get an unequal length of DNA strand. Then used Denaturing PAGE to separate and purify it. I'm only interested to use the forward strand. Any advice if this possible?
I want to extract a protein from polyacrylamide gel to be used for immunization. I am wondering if I need to run PAGE since sds might interfere with the immunization process.
Thank you
Hi all,
I am having keratin hydrolysate in dry form. For performing PAGE which will be the suitable solvent for it as it is not fully dissolving in double-distilled water.
I would also like to know whether any further reaction may occur and interfere with my result (protein is denatured) during the reaction of SDS and mercaptoethanol with my native keratin hydrolysate.
Experts pls give me the answer.
Thanks in advance.
HI,
I am performing the incision and excision assay of 30 mer oligonucleotides of various glycosylases. After the assay, I am resolving them on Urea-PAGE. As far as I understand Urea PAGE resolves only the single stranded DNA but I am getting two different bands. I have also added a control of ssDNA but there are some bands that are quite a bit slower than that. I am confused what is wrong in there. Prior to loading in the gel, I heat the sample with 80% formamide at 90C.
Lane 1= ssDNA
Lane 2= dsDNA with lesion opposite G, No enzyme
lane 3= dsDNA with lesion opposite C,No enzyme
lane 4= dsDNA with lesion opposite A, No enzyme
lane 5= dsDNA with lesion opposite T, No enzyme
Lane 6= dsDNA with lesion opposite G with enzyme
lane 7= dsDNA with lesion opposite C with enzyme
lane 8= dsDNA with lesion opposite A with enzyme
lane 9= dsDNA with lesion opposite T with enzyme
Thanks.
I am willing to Cas13 in vitro. Can it be directly purified from PAGE gel, please suggest me the method.
Currently have punched mouse brain samples of different regions that are of too low protein concentration for Western Blot. Has anyone used the polyacrylamide gel electrophoresis method to detect multiple proteins? Or read it anywhere and can link the literature? Thanks!
I want to run Native PAGE for my G-quadruplex forming Oligonucleotides in presence of a specific metal ion and also to stain it for visualization.Also it is already known that Ethidium Bromide is a weak binder for G-quadruplex. Our lab have Stains-all and Silver stain available. Our lab is new to this topic and no one in our lab has experienced about this.I have tried using some online protocol 2-3 times but it does not work good. Does anyone of you are using this techniques in your lab and if yes, can you please share the protocols for the same.I would be highly obliged.
Samples need denaturing or non-reducing? Should loading buffer contain reduce reagent or/and SDS? Should Gel and Running buffer contain SDS?
My DNA library has 140 nt long sequence with randomization at certain places. We will order the library of 4nmol concentration desalted which has 1.8x10^8 copy number variants. According to the company (from where we are ordering the library), says upon PAGE purification the yield turns out to be 0.5nmol. We want to retain as much as possible from the initial 4nmol, while not losing on the number of variants as well. Does someone deal with DNA library, PAGE purification? If yes, what conditions shall we keep in mind and what percentage of the PAGE is ideal to use?
Hello, I am trying to check how antibody reduction (using 2-MEA) depends on reaction time and analyse the results using PAGE. Following the standard protocol samples should be mixed with sample buffer and boiled before loading, however, temperature activates 2-MEA and the meaning of experiment is lost. I tried loading samples without boiling, but it resulted in additional bands that did not corresponded to MW marker.
Desalting is not an option because of small sample sizes and pricing.
Should I increase the SDS concentration in sample buffer?
Are there any other options?
I am using TBE-Urea gels in PAGE to analyze ssDNA. About half of the protocols (like Qiagen and Biorad) out there insist on pre-running the gel at constant wattage to pre-heat it. However, others (including suppliers like Thermo, invitrogen) do not say anything about pre-running.
Who should I trust and why ? Has anyone actually compared a normal gel to a pre-run gel (ideally for ssDNA) ? Is this dependant on wether you work with RNA or ssDNA ?
I've been having all sorts of issues with my PAGE gels. I just solved the mystery of why they weren't polymerizing, ran one set of gels and they looked great. Second set, after antibody staining, the lanes curve into each other like to the point where they taper and look like everything ran out at a single point at the bottom. What could this be?
Hi everybody,
I would like to ask you, if you have some experiences with these kind of smears - greater than specific products, after dsRNA production. Our template (into in vitro transcription reaction) was purified pcr-products, lenght circa 400 bp with T7 promoter sequences, we used MEGAscript Kit (AM1330) - circa 1ug of template, folowing purification and precipitation with MEGAclear Kit (AM1908).
In the final results, we separated fragments (hoping we got only one specific band) by electrophoresis on 1% agarose gel. I am mostly affraid of too long in vitro transcription - 16 hours on 37°C - but is it even possible to produce so many non-specific fragments? More on, should we use different mass of template into in vitro transcription reaction, or separate final dsRNA on PAGE/denaturing agarose gels?
Have you got some idea, which would explain such results, please?
Thanks for your help.
Matej Medla
Hai. I break the cells containing my expressed protein (r-msAdh1) using non - denaturing lysis buffer. Then I run 10 uL lysate under denaturing polyarylamide gel (SDS-PAGE). But I observed that my protein size is larger than expected (more than 55kD). Expected size is 43kD.
Is it normal? Anyone experience this?
I´m planning to perform Northern blot from Urea PAGE, but I am not sure how to do semi-dry transfer. Any recommendation?
I shall be grateful if anybody suggests me where to do DGGE at affordable charges in India
We are repeatedly troubled with strange Protein bands in PAGE after PCR using Q5 polymerase from NEB. Who has measured the molecular weight of Q5 already and could help us out? Thank you all in advance,
kind regards
Peter
Can the MOPS running and transfer buffers that go with the older Nupage Bis-Tris gels be used with the Bolt gels? If not, does anyone have the recipe for the Bolt running and transfer buffers?
Hi,
I am facing this problem sometimes with PAGE transfer. After I stained with Poncuaue I saw some white horizontal lines on my membrane which are consistent with my sandwich holes. I can understand the air bubbles but I don't know why these holes came up sometimes. Any suggestions? What is the reason behind this? Thanks in advance.
Fellow researchers
I am trying to observe a protein-protein interaction on native protein PAGE (Tris-glycine as a running buffer). The protein of interest interacts with nucleosomes and this interaction was seen as a band shift in native DNA PAGE (Tris Borate EDTA buffer). But, when I ran the same sample on a native protein PAGE, this interaction was not visible as a band shift despite my nucleosomes being intact. I am unable to depict the component (maybe within the gel condition) responsible for the dissociation of this interaction. I am guessing that the interaction strength might be less but not sure what causes its dissociation only in protein PAGE not in DNA PAGE.
Native protein PAGE has resolving layer (pH 8.8) and stacking layer (pH 6.8) made from Tris-Cl, acrylamide, APS, TEMED. The running buffer was Tris-Cl, glycine pH 8.
Native DNA PAGE was made from TBE, acrylamide, APS, TEMED. The running buffer was 1xTBE.
Please provide useful comments or suggestions on this situation.
I am performing PAGE using 15% acryl/bisacryl 19:1 gels with 8M urea. After running my samples, I have to dry the gel to expose it to a X-ray film. First I tried keeping it under vacuum at 70 °C for 2 hours, but the gel broke into pieces when I released the vacuum. The next time I tried to dry 2 gels in parallel: one exactly as I described, another one with the same composition but only 5% polyacrylamide. This time I kept them under the vacuum at r.t. for 3 hours. When I released the vacuum, the 5% gel was perfectly dried, however the 15% gel broke into pieces again!
From this, it is clear to me that high % gels are much more difficult to dry. I would really appreciate if someone could give me advice on how to dry this type of gels. Maybe keeping the gel drying overnight under the vacuum?
We are working on PAGE (Polyacrylamide Gel Electrophoresis) for resolving the amplified DNA samples of wheat crop. But when we tends to prepare PAGE gel, after its solidification, when we remove the rubber gasket the air bubbles fill the whole plate.
Anyone please tell us how can we prevent air bubbles in it?
Thanks.
Hi!
At the upper picture there's an old PAGE gel and at the bottom there's one of the latest from our lab. The latest staining seems to be only half way successful. To both we used phenol/cloroform/isoamyl alcohol plus guanidine thiocyanate/silica methods to extract the viral dsRNA and gels were stained using the silver nitrate method. Both gels are 7,5% polyacrylamide.
What could be the problem? Extraction? Electrophoresis? Staining?
Thank you!
I have seen a number of papers by Kornberg and others using PAGE (Polyacrylamide gel electrophoresis) to separate small (<5 nm) gold nanoparticles by mass. What I am wondering is if there is a way to retrieve the separated particles after PAGE. For example, can you take a poly-disperse mixture of particles, separate them through PAGE, then dissolve the PAGE around just the particles of mass you desire for further analysis.
For reference, the particles I am interested in are very small gold nanoparticles with a thiolate ligand layer, with COOH groups on the ligands providing a net charge for the particles.
Hi,
I have a problem when running polyacrylamide gel electrophoresis (5%) in 1 X TBE buffer with PCR product (starting material 10ng cDNA, 40 amplification cycles). I always got the U shaping band. However, when ran the same PCR product (technical repeat) on the agarose gel (2%) in 1 X TAE buffer, the bands looked fine. The staining protocol for agarose gel and polyacrylamide gel is the same, with GelRed.
Does anyone have the same experience?
Thanks!
Yao
Dear All,
I am trying to standardize Acetic acid/ Urea PAGE electrophoresis for protamine separation. I am able to form stacking and resolving gel. But i am not able to detect any protein on the gel. Also, according to the protocol, it should be run in the reverse polarity. But when i am running the gel sample dye is not entering in the gel.
Please, anybody, suggest the changes.
Thank you in advance...!!
Dear all,
Douglas Pryor Easton
David Farringdon Spencer
Thank you for the valuable suggestion i have run the gel by modifying according to your suggestions. The picture is attached below.Running is fine, the current is dropping till 15mA and proteins are separating.
But this gel I have run without tracking dye.
So can you please suggest which tracking dye will be suitable for protamines?
And I also have paraffine mineral oil for wood) We can buy it later - in 3-4 months but I need it right now( And I am learning IEG and PAGE using Youtube and google so I'm sorry for this stupid question.
Hi, everyone, I meet a problem that I need to run DNA in PAGE gel and limit by next experiment step I can't use TBE as buffer
Can I use TAE buffer instead of TBE?
Is there other method to make PAGE gel to run dsDNA?(low weight , DNA around 50-75bp)
thanks for your reading!
Formamide is necessary to stabilize the single strands of RNA, and is necessary to add in case of rotavirus as i am unable to get bands in PAGE and even i have tried the AGE, i am unable to find a protocol in any research publication that has Full description of the quantities of formamide and other reagents to add in the PAGE procedure. Kindly help.
i want to about the role of these chemicals and why the presence of disulfide makes the environment in the cytoplasm reducing and how
I used Native PAGE to examine the DNA self-assembling nanostructures after annealing. I run my PAGE in a ice bath and 1X TAE was used as buffered system. The gel had been pre-electrophoresised when the samples were added. The gel was stained by EtBr solution after electrophoresis. However, obvious smears emerged in each lane (as shown in the attachment). How can I deal with the smears? Someone have told me that it was normal to have smears on Native PAGE.
Hi gel electrophoresis experts,
I have attached a Ponceau-stained nitrocellulose membrane transferred from a 4%-12% Bis-tris gel with samples that were electrophoresed with a 1x MOPS buffer, 120V constant voltage for 3 hours, with refrigeration. The weak bands on the right look ok, but why are the stronger bands on the left wavy and in one case, the band is narrowed?
Thanks, Mel
Can anyone tell me the usual running conditions (Voltage or power?) for running Urea- PAGE gels?
Should the whole gel running apparatus be used in cold room (6deg) or room temperature? I will be running 8% or 10% Urea PAGE gels and this would be my first time to run such gels.
What voltage of wattage should be used to pre-run the gel and for how long?
Nowadays, famous companies like Merck and Thermo provide different ladders for native-PAGE analysis. However, basic biochemistry tells us that the movement of proteins on the native-PAGE is not only based on the molecular weight but also the PI and shape of proteins, which make them debatable products. Since many research article, even in leading journals like nature, use these products, which encourage others, it is important to discuss if the current native-PAGE ladders are reliable or misleading.
PCR on a DNAzyme for quantification but ended up with mixed negative and positive amplifications. PAGE gel showed proper amplification but why the weird signal?
Dear All,
I have been conducting DNase I footprinting lately, using fluorescence(6FAM)-labelled template.
Would there be a recommended primer purification method for the 6FAM-labelled primers?
The available options are HAP, PAGE, and HPLC. My lab mates and I have been opting for HPLC, but that requires overseas production and takes over a week. So I was hopeful that the other two (HAP or PAGE) might work as well, which would allow me to cut down on the time and the costs.
If anyone has any experience in this and shares their knowledge, it would be very much appreciated. Thank you so much!
Sincerely,
Eun Jung
Every time I make a PAGE gel, the upper stack slightly leaks out of the sides of the cassette and forms bubbles which make the outer two wells completely unusable (see attached image). I've tried making it in the rubber gasket and out of it, as well as with binder clips securing the top, but nothing seems to help it. This is the recipe I've been using: 2 mL 4X upper buffer, 1 mL 30% acrylamide, 5 mL water, 50 uL dye, 50 uL APS and 50 uL TEMED.
Any experience or thoughts on how to fix this?
Hello everyone and thank for your wisdom,
I am trying to detects some plant siRNA by northern blot.
I didn't succeed yet, but I noticed that after dying the gels with EtBr, the migration of my total RNAs is really weird. The migration fronts are getting narrow and moving to the wrong place as if they were being drawn to each others.
do you have any idea of what is happening?
here are some of the main points :
- i use a denaturing polyacrylamide 15% gel (Urea 7Mol. , Polyacr 19:1) and TBE 0,5X as runnig buffer.
- I tried to run my gels either at 80 Volts or at 40V but it didn't changed anything.
- I didn't forget to wash the gel wells with a pipet before loading the samples , to remove the urea deposit .
- The samples loaded are total plant RNA. They were first extracted, dissolved in TE buffer and stored at -20°C one week . They were then mixed in equal volume with a loading buffer containing some formaldehyde , glycerol, and mainly formamide at 50%.
what do you think?
thank you for your time, your advices or your questions
!
and sorry for my bad english, i'm french :P
PAGE is used for various samples. For biochemists, SDS-PAGE is used to separate proteins. My question is about the concentration of PAGE. What is threshold concentration of acrylamide for polymerization?
I am currently optimizing the conjugation of a DNA oligo sequence to a DBCO activated antibody and am looking for a method which could allow me to simultaneously visualize the amount of DNA which has bound to the antibody. Currently, I am able to determine the binding of the DNA to the Ab by visualizing a band shift following coomassie staining on a native PAGE gel. I have also tried spraying the gel with SyBr Gold prior to fixing the gel for coomassie staining in the hopes of doing an overlay, but this hasn't worked! I know that there is a portion of DNA bound to my Ab as I am able to pull the Ab out of solution and when incubating this conjugate with a restriction enzyme and running this on a PAGE gel I can see the DNA. I am wondering if anybody knows of a way that will allow me to visualize the DNA bound to the Ab without having to do the digest as I would like to run downstream experiments with the conjugate?
I have cloned HBV small surface protein in pRSET B expression vector and tried to express it in the E.coli BL21DE3 cells with 1mM iptg conc at 37 deg for 5 hours. After induction i took 3 to 4 ml of culture for from total of 50 ml and centrifuged it at 6000 rpm in microcentrifuge at room temp. Then the pellet from this 3 ml culture was dissolved in 1X Laemmelli buffer(sds gel loading dye). Pellet was mixed by vigourous vortexing and boiled at 100 deg for 10 minutes. then it was centrifuged again at the same rpm and the supernatant(20 ul) was loaded on sds PAGE but still i could not get any expression. My protein of interest is a membrane which has 4 transmembrane domains and has 55 percent hydrophobic amino acids in it. what could be the for no expression. which steps should i follow to express membrane protein. should i run pellet also?
Idont want to transmembrane domains as i need the whole protein.
pleease help me in matlab code for solving the poisson quation in matlab using forth order compact scheme
CHAPMAN & HALL/CRC APPLIED MATHEMATICS
AND NONLINEAR SCIENCE SERIES computational partial d eq PAGE 70
Jichun Li
Yi-Tung Chen
And the question is
page 724 question 3 d part Numerical Analysis
NINTH E D I T I O N
Richard L. Burden
Youngstown State University
J. Douglas Faires
Youngstown State University
We would like to get DNA oligos out of a PAGE gel. Gel cutout fragments would be put into a standard dialysing cellulose tubing (for example 3.5K MWCO) with elution buffer and the filled dialysing tubing is set into a horizontal electrophoretic apparatus so that the oligos stay trapped inside the tubing in the buffer when current is running. Does anyone have experience with such a setup?
During the UREA PAGE prerun at 80V the 0.5M TBE buffer can not reach the required temperature of 60C ? What shall I do ? I have waited more than 1h for the prerun to reach the temperature but it is not working. I am using the BIORAD MINI PROETAN tetra system.
I am trying to analyse proteins qualitatively using sds page laemmli method. Where in this particular case I am using 15% separating gel and 5% resolving gel. The sample loading buffer is as 0.055 M Tris-HCl, pH 6.8, 2 % (wt/vol) SDS, 20 % (vol/vol) glycerol, 4.3 % (vol/vol) β-mercaptoethanol, 0.0025 % (wt/vol) bromophenol blue G-250. On constant voltage setting of 80 volts till dye crosses separating gel (approx 50 min) and then 140 volts till it reaches bottom (approx 2.20 hrs). Due to smile effect and improper resolution of lower marker proteins of ladder, and also broad bands on my unknown sample on my last couple of runs at 12%, I decided to increase gel percentage to 15% and lower the temprature by keeping reservoir tank in an insulated box by adding plenty of ice+water+salt (salt to avoid melting of ice) to submerge 80% of box so as that electrical contacts and top 2 cm of reservoir tank does not comes in contact of the water or ice.
My thoughts regarding this bizarre effects are;
1. If we look up closely on the top of the image their I can find some portion of gel itself wavy, leading to improper differentiating line between separating and loading gel.
2. The temp for run was too low. may be around 0-4C since ice barely melted (not more than 20%). As SDS starts precipitating at lower temperatures below 4C.
3. The vertical dragging of protein marker in extreme last lanes may be due to the air present during polymerisation of separating gel which lead in filling of some of stacking gel therefore lead to it.
4. Do I need to lower the gel% or protein concentration?
Pls note: Ignore the background stain, I stopped destaining as soon as these bizzare bands were visible.
Looking for some explanations along with your suggestions and comments.
Hey guys,
my last couple of Western Blots looked really strange. It seems like the sample goes into the gel pocket more in the middle than at the edge of the lane. when I looked at the transfer using a Ponceau S Staining, the staining was much stronger again at the edges of the lane than in the middle.
I am using a 10% Bolt Bis-Tris gel, precast, from Thermo Fisher. I usually let the gel run at 100 V for about 40 to 60 minutes in MES or MOPS buffer.
I really have no idea what could be the cause for this and if anyone has an idea, I would be really thankful!
I extracted total RNA from bacteria after exposure to lysozyme for 30 minutes (these bacteria are naturally resistant to lysozyme). The 23S/16S rRNA ratio > 2. However, these samples look degraded when I ran polyacrylamide gel electrophoresis (see picture below). What is the reason? Is it a degradation? Is there another explanation?
Thank you for your answer.
Hi,
I want to carry out western blotting for a 233 kDa protein. I was told to use a tris-acetate gel due to the size of the protein. However, I was wandering would I be able to detect the protein if I use a bis-tris gel? As anyone tried this before? If so, what was your experience like?
I am planning on doing Electrophoretic Mobility Shift Assay (EMSA), or gel shift assay, to check the binding of a purified protein to different DNA templates.
Based on a publication that have done the experiment with a very similar protein, I could do the test in a 10% Tris-glycine polyacrylamide gel. However, according to my research, Tris-glycine gels are for protein separation and not DNA. Non-denaturing TBE gels seem to be more appropriate for DNA separation.
My question is, can you use a tris-glycine gel for visualization of DNA ? (I will post-stain the gel with GelRed).
Assume you have a protein heterodimer bound by a single disulphide linkage( monomers sizes 100kDa and 75kDa). How many bands will be observed in a) native PAGE b) Denaturing PAGE? What will be the relative position of bands in each technique?
P.S.: Denaturant used is Urea.
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