Ask questions or discuss issues relating to Gel Electrophoresis.

• Gary Magnant

  Seeking gel electrophoresis tape that is resistant to SDS.

  I am looking for a supplier of pressure sensitive adhesive tape that is suitable for casting polyacrylamide gels containing SDS. Thanks!
• Majid Tafrihi

  cDNA pattern on gel electrophoresis

  Does cDNA have a pattern on gel electrophoresis?
• Tiffany Tan

  Concentrating protein

  I am culturing cells in different conditions. One of the condition gives me very low number of cells so I couldn't detect any bands either for my protein of interest and actin/GAPDH. I wonder ifI am culturing cells in different conditions. One of the condition gives me very low number of cells so I couldn't detect any bands either for my protein of interest and actin/GAPDH. I wonder if anyone has any idea of concentrating protein? I have pooled many samples into one well already =(

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    Tomáš Hluska replied

    I hope my advices are helpful and mainly good :o)

    18 days ago
• Ali Ibrahim

  Is there a software for gel electrophoresis analysis?

  Is there a software for gel electrophoresis Analysis to calculate 1- Number of bands 2- The values of the distance between the bands 3- Draw the phylogenetics tree

  Recent replies ⋅ Show All (7)

  • 
    Eduardo Araujo replied

    Yes fellow, there are some but the free ones are not so effective. Try this: http://www.totallab.com/ or this: http://www.labimage.com.

    Apr 24, 2012
• Bushra Yasin

  Two bands in a gel.

  What does it mean if you get two bands of a protein in your electrophoresis gel instead of one?

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    Christine Longuet replied

    Either phosphorylated/unphosphorylated form, or different state of glycosylation can make a significant shift in apparent molecular weight

    Apr 18, 2012
• Max Francisco Campo

  Cathode and Anode buffer concentration to run a Tricine-SDS-PAGE, 10X?

  I am going to try a Tricine-SDS-PAGE but im not sure if the concentration of the cathode and anode buffers in the chamber is 10X or 1X (Like Laemmli).

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    Joshua Hill replied

    Just plain old 1x

    Apr 17, 2012
• Ju Zhang

  Question regarding protein gel after SDS-PAGE

  I use the 4-20% gradient gel from Pierce. The picture shows the Sypro Ruby staining for whole protein after running. It was generated by a scanner. Naked eye observation on a transilluminator is veryI use the 4-20% gradient gel from Pierce. The picture shows the Sypro Ruby staining for whole protein after running. It was generated by a scanner. Naked eye observation on a transilluminator is very similar. What confused me is that I can see the bands, but they are not distinct from the background and difficult to be compared. The edge of each gel lane has heavy stainning and appears like smear. See the Image. I thought I had a high concerntration of Na+ and K+ in the solution ( both are about ~500mM) ,which may cause the protein aggregate. But after I tried to use choloroform to desalt and purify the protein, I found it gave little help. Anyone had any idea about this problem? P.S.:the two big dots of strong signal at the bottom should be the bluephenol dye, I dont mind too much.

  

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    Hussein Butt replied

    It seems you expected discrete bands, is this correct? If your loaded protein is from tissue, cells etc, then a protein smear is expected as there are numerous proteins that are of a range of sizes.It seems you expected discrete bands, is this correct? If your loaded protein is from tissue, cells etc, then a protein smear is expected as there are numerous proteins that are of a range of sizes. It is very difficult to compare protein bands in whole protein staining unless there is some kind of specific analysis, e.g. immunoblotting with specific antibodies, binding assays/immunoprecipitation, purification.

    Apr 13, 2012
• Padmapriya Kumar

  What is the minimum conc and volume, power in Volts required for detection of a 21mer oligonucleotide in denaturing 16% PAGE by silver staining?

  I want to run the DNA oligonucleotide sequence and stain it by silver staining. unfortunately I donot have a radiolabelled sequence. kindly help me out.

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    Padmapriya Kumar replied

    Thank you very much for the reply.

    Apr 10, 2012
• Cameron Clarke

  Is it possible to prestain with GelGreen /GelRed stains?

  Hello everyone. I'm a newbie here, but am working in a lab and we have a strong interest to use gelgreen as our DNA stain for our electrophoresis. We are trying to see if it can be used as aHello everyone. I'm a newbie here, but am working in a lab and we have a strong interest to use gelgreen as our DNA stain for our electrophoresis. We are trying to see if it can be used as a prestain. We have used it successfully by mixing the stain with the liquid gel prior to pouring (ie in the gel, not the buffer solution), but I was wondering if there is a good way to stain the DNA BEFORE dropping it into the well of the gel. The initial test combining stain and DNA together immediately before insertion into the well, show that the stain does move with the DNA, but it seems to disperse in the gel when voltage is applied. It might be because the stain didn't have time to attach to the DNA. Would combining the stain with the DNA and letting it sit for 15 minutes before insertion into the well make a difference? Is there any way to get the stain to stick better to the DNA?

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    Raúl Reyes replied

    I normally use in the laboratory ethidium bromide, which we all know that until time was of the best ways to get good bands, recently got a sample of GelRed for the staining of my bands, done poorlyI normally use in the laboratory ethidium bromide, which we all know that until time was of the best ways to get good bands, recently got a sample of GelRed for the staining of my bands, done poorly at first, but experiencing and searching on the subject to achieve good results, much higher than with bromide. Another way that occurred to me is to add the PCR microtubes a small amount of GelRed, combining it with the reaction and DYE, so you save time stained and the bands are great.

    Apr 5, 2012

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  • 
    Birendra Singh replied

    Labelled DNA probe (5 fmol)! depends on labelling, added to 10–100 nM of protein (it will be your nuclear extract; concentrate it as suggested by Rajendra) in HGMKE buffer (25 mM HEPES, pH 8, withLabelled DNA probe (5 fmol)! depends on labelling, added to 10–100 nM of protein (it will be your nuclear extract; concentrate it as suggested by Rajendra) in HGMKE buffer (25 mM HEPES, pH 8, with 5 %, v/v, glycerol, 10 mM MgCl2, 20 mM KCl and 0.1 mM EDTA). The mixtures will be incubated at 37 °C for 20 min and separated in 6 % native polyacrylamide gels. The gels will be dried and analysed by a Phosphorimager after a 24 h exposure.

    Apr 4, 2012
• Santosh Kodgire

  Regarding protein re-staining

  I have done Coomassie Brilliant Blue (CBB) staining for protein detection then destained the gel for removal of excess dye not bound to proteins. Now I want to remove the dye bound to proteins to goI have done Coomassie Brilliant Blue (CBB) staining for protein detection then destained the gel for removal of excess dye not bound to proteins. Now I want to remove the dye bound to proteins to go for silver staining with same gel. But in the destaining solution the dye bound to proteins will not remove completely. Could you suggest any other opinions?

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    Santosh Kodgire replied

    Microwave destaining is it will work?

    Apr 1, 2012
• Aruna Sridhar

  GMS stain for Pneumocystis carnii

  My smear is washed off when I use the normal procedure for GMS stain. Can anyone recommend any solutions?

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    Marco Antonio Montes-Cano replied

    Dear Aruna, My work currently focuses on PCR techniques with pneumonia. The GMS technique is used successfully in many laboratories, but also can be used Giemsa techniques. Sorry I did not haveDear Aruna, My work currently focuses on PCR techniques with pneumonia. The GMS technique is used successfully in many laboratories, but also can be used Giemsa techniques. Sorry I did not have much information on this topic

    Mar 28, 2012
• Cilem Purma

  Methods of meat sample preparation for 1D SDS-PAGE?

  Hello everybody, I have meat protein samples prepared according to the protocol as following; 2,5 g of meat sample + 25 ml of Tampon I (tris,sucrose,EDTA) ....centrifugation....(1000g 10 min) Hello everybody, I have meat protein samples prepared according to the protocol as following; 2,5 g of meat sample + 25 ml of Tampon I (tris,sucrose,EDTA) ....centrifugation....(1000g 10 min) ...trowing of supernatant... precipitated material + 25 ml of Tampon II (Tris, EDTA) ....centrifugation....(1000g 10 min) ...trowing of supernatant... precipitated material + 25 ml of KCl (0,15 M) ....centrifugation....(1000g 10 min) ...trowing of supernatant... precipitated material + 25 ml of KCl (0,15 M) ....centrifugation....(1000g 10 min) ...trowing of supernatant... lyofilization of precipitated material. Is it possible to use the same sample preperation protocol as 2D SDS-PAGE to prepare the liyofilized samples for loading into the wells as described below? And after the last step what do I do to make the samples ready to load on sds-page gels? ...weight 0,03 g of lyofilized sample in a 2 ml eppis 500 microliter lysisbuffer 32,5 microliter DDT 20 microliter phosphatase inhibitor ...wait 30 min in ice ...ultrasonic bath for 1,5 min. ...wait 10 min in the ice ...centrifuge at 20.000 g-4C-30 min ...collect supernatant in a 1,5 ml eppis ...use bradford to determine protein concentration ....?????? Or is it better to use the protocol in which we use laemmli sample buffer + beta-mercaptoethanol and water bath at 50 C for 1 h overnight before filtering the solution and loading the sample directly into the wells with 7 microliters of bromofenol blue?

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    Gladys Thalia Cortés replied

    If you use 10% 2ME, it is for reducting conditions of your sample, looking for conformational epitopes. If you use 1D-SDS-PAGE and 2ME Laembli buffer conditions in SDS-PAGE used for proteinIf you use 10% 2ME, it is for reducting conditions of your sample, looking for conformational epitopes. If you use 1D-SDS-PAGE and 2ME Laembli buffer conditions in SDS-PAGE used for protein desnaturalization. You can see Molecular & Biochemical Parasitology 129 (2003) 127–135 Characterization of proteins localized to a subcellular compartment associated with an alternate secretory pathway of the malaria parasite Gladys Thalia Cortes a,∗, Enrique Winograd a,1, Mark F. Wiser b

    Mar 27, 2012
• Ashfaq Ahmad

  http://expertscolumn.com/content/silver-staining-protocol-stain-sds-page Protocol of silver staining of SDS-PAGE gels, I used it personally and it worked very well. Silver staining is much morehttp://expertscolumn.com/content/silver-staining-protocol-stain-sds-page Protocol of silver staining of SDS-PAGE gels, I used it personally and it worked very well. Silver staining is much more (1000X) sensitive than the normal ordinary commasive blue staining. Thanks
• Israr Ahmad

  What is the role of PVP in CTAB buffer?

  What is the role of beta-mercuptoethanol?

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  • 
    Vikrant Piprode replied

    hi israr....mercaptoethanol is basically a reducing agent that breaks the disulphide bonds in protein when running a reducing SDS PAGE electrophoresis......so if you have a protein with more than 1hi israr....mercaptoethanol is basically a reducing agent that breaks the disulphide bonds in protein when running a reducing SDS PAGE electrophoresis......so if you have a protein with more than 1 subunit liked by disulphide bonds this agent can be used to find out the no. of subunits in a protein.

    Mar 25, 2012