Gel Electrophoresis

Gel Electrophoresis

  • Ravi Cholia added an answer:
    How can I avoid slimy total cell lysate extraction?

    I am facing a problem during extraction of total proteins from cell lysate. I am using cell lysis (RIPA) buffer with standard composition as described in scientific literature. But every time my samples goes slimy which creates trouble for me, for my next experimentation.
    If someone have solution regarding to this problem, please share with me.

    Ravi Cholia · Central University of Punjab

    Thanks Mr Gary James Spencer..... I will try this also. Thanks to all for giving their valuable time and suggestion to my query.

  • Yoann Pageaud added an answer:
    What is the minimum DNA quantity (ng) required to visualize DNA on Agarose gel?

    I have very low amount of DNA 10 ng/ul. So if I would like to check on gel, how much ul I have to take?

    Yoann Pageaud · Université d'Évry-Val-d'Essonne

    Yes totally depends of the concentration of agarose in your gel, and how much BET you use. I don't if a way to improve the incorporation of BET in DNA exist, try look for it.
     

  • Olivier Monestier added an answer:
    Why can't I get positive results for the expression of the Myostatin gene in cell lines developed from muscle?

    In the initial try, I couldn't get positive results what may be the reason?

    Myostatin inhibit the proliferation and differentiation of muscle cells.

    In proliferation stage, the expression level increase with the cells confluence (inducing the end of proliferation by CDK2 inhibition).

    In differentiation stage, the myostatin expression also increase with a climax  after 60 to 70 hours (if i remember well).

    So, the expression of myostatin is barely detectable during the first days.

    I hope this will help.

  • Khaled Alkharsah added an answer:
    Should genomic bacterial DNA migrate in the gel even if it is not digested?

    Hi,

    I am trying to characterize some MRSA strains with PFGE. The DNA marker (SC DNA ladder or Lambda DNA ladder) migrates nicely through the agarose gel. The bacterial DNA on the other hand does not. I see the DNA stacked in the well. I am not sure whether the bacterial genomic DNA is digested or not. I tried several protocols but were not successful. Any recommendations? Should the genomic bacterial DNA migrate in the gel even if it is not digested?

    Khaled Alkharsah · University of Dammam

    Hi Sandra,

    Thank you for your help. Actually I also do the same. but the problem is that at the end i get the DNA stucked in the wel of the gel while the ladder migrates witout problems.

    I have a look at the protocol you sent. may i 'll find a trick which helps.

    regards,

    Khaled 

  • Michael B LoMonaco added an answer:
    Can anyone help with SDS-PAGE protocol recipes problems?

    Hello there, i have a problem with an SDS-PAGE protocol recipes. the gel did not polymerized except when i increased the APS concentration and the TEMED volume, but when i run the samples in this gel they didn't move at all, how can I solve this problem and if anyone can suggest a working protocol. thanks a lot.   

    my protocol actually worked previously but this time the samples moves very slowly in the gel ( stacking or the resolving the same), is it possible that my power supply give false voltage numbers ?? thank you all

    Michael B LoMonaco · University of Rochester

    Also, acrylamide breaks down to acrylic acid and TEMED to ammonia.  Dowex ion exchange resin can remove acrylate ions if you must make your own solutions. 

  • Ann Samarakkody added an answer:
    Can anyone please help with native polyacrylamide gel streaking?

    I have ~ 150 bp long dsDNA and I ran them in a native polyacrylamide gel (8%, 29:1 in TBE).

    I keep getting these weird streaking bands. Any suggestions? 

    Ann Samarakkody · University of North Dakota

    1. If you suspect it's your buffer, you could try the commercially available 6X buffer or simply make your own. (I usually use the 6X loading buffer

    2. If you suspect it's your samples/marker gone bad you could try the same samples on a agarose. In addition try another marker. Since you are troubleshooting may be try some freshly prepared DNA from some extra cells?

    3. If you think it's your acylamide gone bad you could try the precasted gels that are commercially available. In addition i would try pouring a 6% gel since the acrylamide percentage is 29:1

    Good luck!

  • Brandy Wade added an answer:
    Strange smuding and too much protein on my western blot, does anyone know why this is happening?
    I loaded 7.5ug of protein extracted from mouse spinal cord lysate in each lane but after doing a ponceau stain and seeing significant smudging, I have been led to believe that this is too much. This is weird to me because I have loaded 30ug of mouse proteins before with no problems. Is too much protein definitely the problem? I have repeated this twice with different samples and have had the same problem. The gel used was SDS-Page, Nupage 4-12% bis-tris 1.5mm and 10 well gel.
    Brandy Wade · Emory University/ VA

    Try sonicating your samples after heating to reduce the smearing of the large "blob". I have used these types of gels for brain homogenates and sonicating the sample helps give better resolution of proteins (especially when you have some much of it).

  • Dina Morshedi added an answer:
    Does anyone purify recombinant alpha synuclein?

    When I look at the SDS-page pattern of alpha synuclein in some studies its band appears higher (around 17 KD) than the real place of its molecular weight (14.4 KD). However, our purified protein appears at around 15 KD. The sequence is OK and N-terminal also was analyzed and it is OK too. Dr.Sidhu in "Abnormal migration of human wild-type -synuclein upon gel electrophoresis" showed that ASN migrated in SDS-PAGE unusually, so why would our protein migrate and appear in its real position?

    Dina Morshedi · National Institute of Genetic Engineering and Biotechnology

    Thank you Sleman. In all reports it was expressed in E.coli which has not high PTM activity

  • Syed A Morshed added an answer:
    How can I explain a non-specific band on a western blot that appears with several different probings of different species?

    I have been reprobing the same two blots most of the week and keep noticing a band in the 50-75kD range that doesn't correspond to anything I'm probing for. What's even more odd is that it appears when I probe with mouse monoclonal or rabbit polyclonal and bovine-anti-mouse or goat-anti-rabbit, respectively. Can anyone explain how that's possible?

    Syed A Morshed · Mount Sinai School of Medicine

    It depends on antibody types. I have been characterizing both monoclonal and polyclonal antibodies from human, mouse and hamster. Linear/continuous/sequential and conformational/native/3D epitopes are critical for antibody binding. WB is unable to detect the 3D one, even native gel may fail if the directed epitope has some protein modification and same goes to ELISA, however, it depends on fixation methods for ELISA. To detect 3D binding, best would be IP and needs confirmation with a linear antibody. Unfixed fresh frozen tissue section (5micron) is a good for conformational one. Another choice is MALDI-TOF/MS-MS for the 3D type. For non-specific bands on SDS-PAGE gel with different species, you need to do absorption assays to see the reduced level of binding. For receptor or extracellular ligand molecules, you can run FACS. So one assay cannot give you the final or confirmatory answer for the nonspecific reactivities. Also keep in mind that concentration of both antigen and antibody is an important regulator for proper binding as they may impose prozone phenomenon. Thanks.

  • Michael B LoMonaco added an answer:
    Are there any methods to amplify unknown proteins using PCR?

    We are using beetles within our experiment and having a difficult time extracting enough protein to obtain a good gel electrophoresis result. I am wondering if there is a simple process using PCR to obtain more of these unknown proteins? Or any other advice would be great! Thank you in advance. 

    Michael B LoMonaco · University of Rochester

    Further, it is possible to use PCR based immunological detection of protein but I have no direct experience nor can I attest to its linearity.

  • Cedric Hurth added an answer:
    Does anybody know if there are any commercially available replaceable gels for capillary gel electrophoresis?
    I would require some commercially replaceable gels for capillary gel electrophoresis
    Cedric Hurth · The University of Arizona

    I am assuming you are trying to separate DNA via CE.

    In recent years, the work of Annelise Barron at Stanford has shown the efficiency of mixed pDMA (polydimethylacrylamide) and linear polyacrylamide. But the synthesis of these methylated acrylamide may be tedious.

    I personally chose to use a mixture of hydroxyethylcellulose (HEC) and polyvinylpyrollidone (PVP) since 2009, after collaborating with Bruce McCord at Florida Internatioanl - who developed the system. The solutions can simply be prepared from commercial powders obtained from Sigma (HEC: 250 kDa | PVP: 1 MDa) while adjusting the proportions and completing with urea and, for instance, TAPS buffer.

    For more information, download my 2012 and 2010 papers in Electrophoresis from my profile.  References for Bruce's initial work are contained therein.

  • Michael B LoMonaco added an answer:
    Can anyone help me resolve a 10% separating gel, for PAGE, surface issue ?

    Hi guys,

    So while carrying out PAGE, following the addition of propanol alcohol, the surface becomes smooth, but not straight, it usually starts to take a lower angle near the end. It's not a huge deviation from the straight line, but it is noticeable when working. Is it an issue I should be concerned about? Could it affect the electrophoresis? It happened more than one time by the way, and hence my question. Many thanks for your time.

    Michael B LoMonaco · University of Rochester

    Moe

    Find a leveling stand.  It could be that your bench is not 180.

  • Velu Mani Selvaraj added an answer:
    How do I reduce smear on a qRT-PCR melt curve?
    I obtained this melt curve from qRT-PCR (one Step RT-PCR). The peak is so broad and when I run the gel, there was primer dimer smear below the desired product. How do I reduce this primer dimer smear and get a sharp peak?
    Velu Mani Selvaraj · University of Hyderabad

    I still suspect the bands that you observe are not Primer Dimers but non-specific ampification. Consider that your primers are 25 bases each and so from end-to-end they make utmost 50 bp if they form PD, which is still a stretch. PD length would be less than the combined length of the individual primers. It is apparent from your gel picture that the bands are above 50bp. Probably your primers amplify isoforms. As Amanda O'Donnel mentioned earlier, you need to sift the literature for any isoforms known and design primers to be specific for your target. Also, you can sequence the non-specific band that is amplifying and that should help resolve your problem.

  • Siti Ramlah Ahmad Ali added an answer:
    Does anyone have an experience to handle smiling gels?

    Smiling of bands near the edges of DGGE gels appears to be endemic in all systems. What is the exact cause of this?

    Siti Ramlah Ahmad Ali · Malaysian Palm Oil Board

    Leakage, so never use the last well at both ends. It is best to balance the loading & leave the last 2 wells at the two end vacant

  • Amrita Sinha added an answer:
    I am working on study of genetic diversity using SSR. Can anyone kindly tell me how to prepare an input file for the software ARLEQUIN?

    I am using horizontal gel electrophoresis for my work.

    Amrita Sinha · Indian Council of Forestry Research and Education (ICFRE)

    Thank you all for your response. 

  • Dipankar Ash added an answer:
    Can SDS PAGE gels be saved after running?

    Is it possible to store an SDS PAGE gel to transfer the next day?  I have seen some people say it can be stored in 1% acetic acid at 4 degrees, others say just water.  

    Does anyone have experience with this?

    Thanks!

    ~Anna

    Dipankar Ash · National Institute of Immunology

    After completion of gel run , the gel can be stored in transfer buffer which you are going to use for transfer at 4 degree for over night. In fact gels should be kept in transfer buffer for 1 hr before setting transfer for WB. 

  • Pulu Sun added an answer:
    Does anyone know why there is a partial smear in this SDS PAGE? (picture attached)

    I had problem with one of my protein sample but not on the other protein (similar protein but from different plants).
    There was smear on the lower part of the gel (see attached picture).
    I repeated the same experiment for twice and both had the same problem.
    Can anyone explain why and how to solve this problem?

    Thank you very much!

    Some parameter for the gel:
    1. Denaturating polyarylamide gel
    2. Acrylamide 10%
    3. size of protein (16 kDa)

    -------------------------------------------------------------------------

    Edited: 05.09.2014

    Thank you so much for all the suggestions.

    I have tried to increase the content of acrylamide from 10% to 15%, but it did not work. I also tried to extend the incubation time also increase the incubation temperature. However, the smear still existed. At the end, I realized that the smear was caused by the function of the protein itself...the protein has high hydrolysis property....I think this is the reason why there was smear during electrophoresis.

    Pulu Sun · Université Jean Monnet

    Thank you very much for all the suggestions.

    I have tried to increase the content of acrylamide from 10% to 15%, but it did not work. The smear still existed. At the end, I realized that the smear was caused by the function of the protein itself...

  • Ann Holtz-Morris added an answer:
    Is there a technical reason why if I loaded double the protein concentration I see less signal of my target protein, but actin looks similar?

    So, I ran a gel looking at a low expressed protein across many samples with about 25 ug of protein. My actin looked fine when it came out, but I had very low signal except in my positive control. So, I doubled the protein and ran the samples again and this time I saw even less signal on the gel, but even the background signal was cleaner than my previous attempt and even my positive control was not detectable, but the loading looked fine. I didn't change my transfer conditions, but maybe there was something off with my antibody dilution. Any suggestions?

    Ann Holtz-Morris · Children's Hospital Oakland Research Institute

    For reasons similar to Jurgen's, I prefer to run gradient gels. Yes, they cost a little more, but the resolution makes up for it. (Unless you've poured a LOT of gradient gels, they're really hard to replicate so just buy the commercial ones.)

  • Pablo Bolaños-Villegas added an answer:
    Does anyone have experience on COMET assays?

    Does anyone have a good protocol for COMET assay for plant root tissue? 

    Pablo Bolaños-Villegas · University of Costa Rica

    Dear Preetam, I am sharing with you a bench protocol, a protocol for the preparation of buffers, the user´s manual for the comet assay kit from TREVIGEN, a fact sheet for Bleomycin sulfate, plus three papers that might be useful. I many need your e-mail address to send you the software for scoring comets. Best regards, 
    Pablo

  • Nosrihah Ismail added an answer:
    Which kit is more accurate for determination of protein concentration in SDS- Page sample buffer?

    Which kit is more accurate for determination of protein concentration in SDS- Page sample buffer?
    1- Protein assay kit (Bio-RAD)
    2- 660 nm protein assay (Thermo scientific)
    3- 2-D quant kit (GE healthcare life science)
    Protein is extracted from lung biopsy and finally western blot will perform

    Thanks

    Nosrihah Ismail · University of Kuala Lumpur

    2D Quant kit compatible with SDS buffer.

  • Michael B LoMonaco added an answer:
    Can anyone help with a question about Post Bradford Assays and Protein PAGE?

    Hi guys, hope all is well,

    so I just finished bradford assay on three different cell lysates. The chosen protein concentrations after analyzing the results were 3.5362 ug/ul, 0.6579 ug/ul, and 2.8289 ug/ul. My question here is when preparing the samples for loading prior to gel electrophoresis, what criteria should I consider in order to determine protein concentration in wells, sample volume in each well, ... etc. Listing examples would be very greatly appreciated. Many thanks for taking the time to read my question.

    Michael B LoMonaco · University of Rochester

    If it is not clear to you, each sample should contain the same volume/ratio of loading buffer.

    Does anyone disagree with this assertion?

  • Toni Leigh Goldswain added an answer:
    Can GAPDH be used as a loading control?

    Good day

    I was told a few weeks back that GAPDH cannot be used as a loading control in differentiated cells since its expression does not stay constant. Has anybody heard anything about this?

    Toni Leigh Goldswain · Stellenbosch University

    Thank you all for the replies. I received an article from the company we bought the antibody from so I'll go by that. I will be sure to use more than one loading control in future, but for now I am finished in the lab. 

  • Steingrimur Stefansson added an answer:
    How do I check oligomers / dimers of the protein using Native Gel ?

    I am new to protein chemistry,

    Can I know how do I check oligomers / dimers of the protein using Native Gel ? Thanks in advance.

    Steingrimur Stefansson · HeMemics Biotechnologies Inc

    Hi, I agree with Dylan. Size exclusion chromatography only separates proteins based on size (if your protein is globular). Native PAGE separates proteins based on size and charge.

  • Negative control give a "ladder like band" on gel electrophoresis when using OmniAmp LAMP reaction kit. How to troubleshoot the LAMP reaction?

    I'm a postgraduate student and my research is about molecular detection of bacteria by using LAMP method. Because in my country, Malaysia the cheapest kit for LAMP was from Lucigen (OmniAMp LAMP reaction kit), therefore I immediately purchase 500rxns of LAMP kit. However, after a few month of trials and optimizing the reaction mixtures, the result always give a band formation for negative control reaction.

    But when I try to used my colleague LAMP kit from Eiken (LoopAmp LAMP kit), the positive samples (confirmed with PCR) and negative control give an expected result and no band in negative control.

    What could be the problems? Is there any other suggestion, how to troubleshoot this matter?

    Javier Gandasegui Arahuetes · Universidad de Salamanca

    And could be dimer primers also

  • Roland Iosif Moraru added an answer:
    What is the best safety glasses to use within a bio lab?

    Anyone to suggest a good safety glass brand or a model to use in a biological lab. I found couples of UVEX glasses, but I'm not sure they're appropriate to use in a bio lab. They actually do prevent 99.9% UV which I consider as a feature (I'll be working with UV and gel electrophoresis). 

    Roland Iosif Moraru · University of Petrosani

    I am sorry, being in hurry I forget the link.

  • Attila Lehotzky added an answer:
    Anybody have any experience looking for human EGF in western blots?

    I have been trying to identify analogues to human growth factors in biological secretions. I am having trouble detecting EGF, even in the positive control. I have tried increasing the gel to a 20% gel, decreasing the transfer time to 45 minutes and using 2 PVDF membranes to try and catch the EGF if it manages to transfer through the first sheet. We are using reducing western blots and running at 60V through the resolving gel and 40V through the stacking gel until the loading front is about 2/3 the way down the gel. It may be the case that our positive control is simply not working, but before I try and repeat it, I would like to know if there are any modifications that should be made to the western blot method when looking for small 6 kDa proteins?

    Attila Lehotzky · Hungarian Academy of Sciences

    Small peptides retention on blot could be improved cross-linking by FA or GA just after blotting. It might be help, if did not disturb the detection..

  • M.Ahil Kannan added an answer:
    How much of my purified protein is necessary to perform 2D-PAGE?
    My sample is a purified protein and I need a two-dimensional electrophoresis to show at least two isoforms. The strips I can use are pH 3-10, 7cm or 17cm. I don't know the protein quantity I have to use. My group doesn't have experience with purified proteins, but we thought 10ug on both strips are sufficient. What do you think?
    M.Ahil Kannan · Bharathidasan University

    My answer is same what you assumed. where you going to use crude protein there is may be need to study all the proteins expression. But in your case, you have pure protein, the concentration is enough for both silver and coomassie staining.

  • Bharat Vaidyanathan added an answer:
    Does Sigmas skim milk powder contain free biotin?

    I am working with streptavidin coated beads and as a part of my protocol I have to block the beads with 2% skim milk buffer. Well, it has brought to my attention that some of the milk powder available might contain free biotin. Unfortunately, Sigma has never tested for biotin in their products.

    Does anybody know whether skim milk powder contains free biotin or not?

    Bharat Vaidyanathan · Memorial Sloan-Kettering Cancer Center

    milk is not preferable for Streptavidin due to biotin. You can try 10% Casein, which is biotin free.

  • Samra Sardar added an answer:
    Which method is good to quantify the protein concentration tissue lysate before loading in well for western blotting?

    Determination of protein concentration is very important befor loading the samples in wells. So, can you please tell me the most effective and easy method by which protein can be quantified in tissue lysate.

    Is there any interference of proteases inhibitors or lysis buffer contents on the determination of protein content by any method. As earlier i experienced the problem in determination of protein content in tissue lysate in lysis buffer + protease inhibitors by Lowery method. 

    If can you give me the full method of determination of protein content starting from tissue collection then its lysis and then determination.

    Samra Sardar · University of Copenhagen

    Dear Vinay, I use protease and phophatase inhibitor tablets from Roche. I can send you the product codes, if you want. The volume of RIPA (or any other tissue) lysis buffer depends on the weight of tissue. I usually use 300ul for half of mouse spleen

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.

Topic Followers (18757) See all