Protein Marker Related Questions


1. Gel with 15% acrylamide in Tris-Glycine system is usually able to well separate small proteins ranging from 5 to 10 kDa.

2. Gel with higher than 15% acrylamide can be used, however, less satisfied resolution of protein separation is frequently observed due to technical difficulty in preparation of gel higher than 15%, including uneven polymerization, bubble formation, and most importantly, the ambiguous results for the small proteins/ peptides. If gel with higher than 15% acrylamide is needed to be used, we suggest:

a. Minimize the length of stacking gel. The long length of stacking gel will lead to insufficient stacking of very small proteins, and thus causing ambiguous result. Less than 5 mm (from the well bottom to the top of separation layer) of stacking layer is suggested.

b. Adjust voltage and time of electrophoresis. To obtain better stacking of small   proteins, 100V/15min followed by 150V/ 60min is worthy of try when running with a 20% TG gel.

c. Use loading tip.  When loading the protein marker or the protein samples, we suggest using loading tip which can easily get close to the well bottom and therefore minimize the problem of insufficient stacking of very small proteins.

3. Try using a Tricine gel. Tricine gels are ideal for separation of peptides and small proteins with a molecular weight <10 kDa. Superior resolution is achieved by slowing the migration rate of the peptide-SDS complexes. This helps achieve separation from the faster-moving SDS micelles that interfere with peptide   resolution in Tris-glycine buffer systems.

Here are possible causes and solutions

A. May be masked by the prestained protein: extend the electrophoresis time to separate prestained protein from the low molecular weight bands.

B. Not suitable transfer membrane: try to use 0.22 μm PVDF.

C. Inappropriate methanol concentration: 

(a) Use freshly prepared transfer buffer. 

(b) Try to increasing the methanol concentration in the transfer buffer (up to 15% ~ 20% v/v methanol).