Hello Mervi,

Unless your gels are of poor quality, there should be no problem in distinguishing two polypeptides with Mw 51 and 55 kDa by SDS-PAGE analysis. I would use a regular Lämmli gel with 12.5 % polyacrylamide, avoiding to overload samples (ideally no more than 1 µg/band, run several different loadings if need be) and run the samples next to each other, if possible with a control that you know contains the two species. That should save you a lot of hassle compared to 2-D gels.

Good luck,

Pierre

Hi Mervi T Hyvönen, there is one way by which you could differentiate between such protein isoforms is by performing 2D- gel electrophoresis (separates isoforms of proteins based on their molecular weight and isoelectric point (PI)).

Hello Mervi,

Unless your gels are of poor quality, there should be no problem in distinguishing two polypeptides with Mw 51 and 55 kDa by SDS-PAGE analysis. I would use a regular Lämmli gel with 12.5 % polyacrylamide, avoiding to overload samples (ideally no more than 1 µg/band, run several different loadings if need be) and run the samples next to each other, if possible with a control that you know contains the two species. That should save you a lot of hassle compared to 2-D gels.

Good luck,

Pierre

I would add a lane in which you run a mixture of your samples. The difference in molecular weight may not be enough to see differences between adjacent lanes easily by naked. But if you get a double-band with your mixture, you have at least a control that there are different sized molecules present.

You could then analyze the lanes closer from a scan with your compurter.

2-D gels are useful for proteomic analysis more than differentiationg two different isoforms. Have you ever thought about capillary electrophoresis? https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Supplemental_Modules_(Analytical_Chemistry)/Instrumental_Analysis/Capillary_Electrophoresis

You can perform 2-D gel electrophoresis...

Thank you all for your answers. I will first try with 1D PAGE, and if not working, then with 2D.. If my antibody is not very specific, then other problems may arise with 2D.

Would it be better to run on 18% gel for longer time, does it separate better than ordinary 12% gel?

The 12% is likely sufficient, but I would try an 18% gel, too.

A shallow gradient gel (say, 10-15% T) results in narrower bands and hence better resolution.

Alternatively, you could use a transverse gradient to identify the %T for optimal resolution (doi:10.1016/0003-2697(88)90581-7).

Companies that sell pre-cast gels usually show migration charts of gels with protein ladders. I would look at those when selecting percent acrylamide, and whether a gradient gel might work. For example,

http://www.bio-rad.com/en-us/product/mini-protean-tgx-precast-gels?ID=N3GRW04VY

for the specific type and size of pre-cast gel sold in the above link, the distances between the 50 and 75 kd protein standards, as well as between 50 and 35 kD standards are largest. I would then either use that pre-cast gel or use that information as a guide when preparing the gel. This would apply to a 1-D or the second dimension of a 2-D gel.

Amended on 6 July 2019

I must sadly say that notorious SDS-PAGE can not be utilized in Protein Biocheistry or Proteomics. Poly acrylamide gel (PAG) is representative hydrophobic material (please see file; IEF for hydrophobic protein).

Further, capillary electrophoresis (CE) is not good for separating human serum biotinidase (relatively hydrophobic glycoprotein; please see file, "Dr. Terentyeva Urine BIN") (our unpublished observation studied with Dr. Franco Tagliaro (University of Verona, Verona, Italy)). Interaction between hydrophobic proteins seemed to be large. Therefore, separation method using HPLC must have been developed by us.

Separation of proteins should utilize the high-recovery RP-HPLC method of us (please see file; Lysozyme by RP-HPLC). Proteins are separated in accordance with hydrphobic interaction; i.e., Hydrophobicity and Relative Molecular Mass (Mr). Therefore, RP-HPLC column for protein separation is short due to gradient elution, and the particle diameter of ODS silica gel seems to be optimum at 10 μm (or 10,000 nm or 0.01 mm). ODS silica particles are resistant to mechanical shock by utilizing the silica gels with 5.0 and/or 10 μm particle size (my unpublished finding at Shimadzu Co.).

Separation power by high-recovery HPLC-SEC method seems to be weaker than RP-HPLC method (please see file; SEC column 300A silica).

Hydrophobicty (%) is defined as follows; i.e., Gly x 0.5 + (Cys + Met) x 1.5 + Ala + Ile + Leu + Val + Pro + Phe + Tyr + Trp (%).

Hydrphobic membrane proteins and/or Peptide/Protein hormone receptors are defined as the proteins possessing larger values than 52.5%.

Glyco-chain structure is also important in Glycoprotein separation; i.e., Sialylated or N-Acetylneuraminic acid (NANA) glycoprotein elutes faster than Asialo-glycoprotein (my unpublished observation).

Silica gel seems to interact to cysteine residues of proteins, and please defend the Cys residues by Pyridyl-Ethylation (PE) (please see file; PTH Roma RP-HPLC).