I work on highly positive charge short hydrophobic peptide production from an E. coli cell lysate, M.wt. 6.5 KDa. I used 16% SDS-PAGE with 0.376 M tris HCl as separating gel and 0.025 M tris HCl as stacking one, all i get is this gel profile.
The last band in the marker is about 6 KDa.
I see that sort of "squeezing in" of the gel lanes when my samples have high salt in them, usually diluting them a bit further with loading dye will make the lanes run better.
If you need them undiluted, however, you can perform dialysis to remove the excess salts. Best of luck!
Additional Silver is right, for low MW is better use appropriate tools,
1. Tris-Glycine page
2. Marker or ladder specific for peptide
Good luck
Hi Amira, I have been running SDS-PAGE gels for decades and I have to admit that I don't remember seeing a "bullet" lane like this before.
I don't think this is due to high salt because that would make all the bands blurry and not run right. Yet you have great looking bands until the sample comes to a point at the end.
Please share with us the buffers and conditions you use.
Amended on 6 August 2018 (The day when we have been dropped with Atomic Bomb on Hiroshima in 1945)
I must sadly and repeatedly say that SDS-PAGE is not work on Hydrophobic proteins and peptides (please see file; IEF for Hydrophobic protein). Poly-acrylamide is a hydrophobic material only applicable to hydrophilic molecules such as Nucleic acids (RNA and DNA). Hydophobic interaction as first described by the Dutchman physicist Dr. van der Waals is unexpectedly strong. SDS (sodium dodecyl-sulphate; with C12 fatty acyl chain) is not sufficient to prevent the van der Waals' force. Sodium palmitoyl sulphate, if available, may be desirable.
Effect of salt is present, and I have used Weber-Osborn's phosphate buffer system instead of notorious Laemmli's Tris buffer system (please see file; Resistance to Hg), but Weber-Osborn' method is still not perfect.
The reason of effect is not known yet, human pancreatic juices can only be separated by the method of Weber and Osborn; i.e., the method of Laemmli is not possible to apply to the pancreatic juice (please see Fig. 5 of the file; HPLC-Surf-SEC protein determination method). I am now very regretting for not had mentioned this method-differences in the pancreatic-analysis in that report.
Therefore, I have previously developed the reliable high-recovery RP-HPLC method (please see file; Lysozyme by RP-HPLC).
Further, I also have developed an HPLC-Surf-SEC protein separation method (please see file; SEC column 300A silica). The importance of high-recovery analysis in HPLC has reported in Verona, Italy (please see file in Japanese; Verona Report). In the case of linear molecule of sulphated-poly-L-fucose (fucoidan), 300 A silica can exclude fucoidan (Mr 200,000) at the position of V0 (column void volume), most proteins and/or peptides are non-linear or globular molecules to be excluded by narrower pore-sized silica-gel-column. 50 A silica gel can exclude peptides of Mr larger than 500 (please see Fig. 1 of the file again; HPLC-Surf-SEC protein determination method). Therefore, Mr 6,500 peptide should be separated by using 100 A Diol-type silica gel column.
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