Resolving LMW proteins

Eric Hebert erich at
Tue Mar 29 21:20:00 EST 1994

In article <ming-280394223916 at>, ming at (Ding Ming) writes...
>In article <1994Mar28.180207.1 at>, ajayaram at
>> Hi there,
>> I'm trying to run a 8 kd protein on a SDS get but I'm having some problems
>> getting it to show up due to its small size.  I've tried 10, 12 and 15
>> percent gels but the but the protein doesn't seem to be there ( the problem is
>> obscured by the fact this protein is being made after an induction and the coli 
>> proteins interfere with seeing the bottom of the lane where the 8 kd protein
>> would presumably run.)  I've tried running the gels without loading dye in the
>> sample buffer thinking that the dye front would obscure the 8 kD band.  This
>> didn't help.  If anyone has any suggestions about resolving low molecular
>> weight proteins please respond by posting in this group or emailing me
>> directly.  Thanks.
>> Arul
>> ajayaram at
>Dear Arul,
>You should be able to see the 8kd band very easily. I am working with
>proteins of 3.5 to 6.5kd and they behave very well on SDS gel. Two points
>for small protein sds-page:
>1)Some small proteins are not easily detected by Coomasie Brilliant Blue
>and therefore, you have to use different staining methods;
>2)If the protein didn't resolve well on a classic Laemmeli
>system(Tris-Glycine)(usually 15% to 18% gel should work for 8Kd protein),
>you should try Tricine buffer system if you havn't done that. For detailed
>procedure about the tricine buffer system, please refers to Anal. Biochem.
>166:368-79. Good luck!
>Ding Ming
>University of Wisconsin-Madison
>Telephone: (608)265-3544     Fax: (608)262-7420

As a followup to the resolution of low molecular weight proteins on SDS gels.
I agree fully with Ming on the use of the Tricine gels stated in the above
reply, however, the resolution of protein below 6.5 kD using the normal
Laemmeli procedure is difficult, since stacking is prohibited by the fact that
the proteins do not separate from the bulk SDS front.  From my experience,
I would strongly recommend using the Tricine based gels instead of pushing 
the Laemmeli gels to percentages greater than 12-15%.

The second problem touched briefly by Ming's reply deals with the detection of
small molecular weight fragments or protein using Coomassie Brilliant Blue (R).
I am unsure of the detection limit or the binding efficiency of small peptides
to CBB(R) however, another cosideration may be your fixing step.  The standard
methanol fixing and staining solutions used may cause an increased solubility
for the peptides.  I have utilized a different staining technique, which I 
believe was developed by David Goldenberg (U of Utah) for his work on BPTI and
BPTI fragments.  

Staining Solution
50 g Trichloroacetic acid
50 g sulfosalicylic acid
0.5 g CBB (R) - must be electrophoretic grade
dissolve in 500 ml of dH2O, and the stain can be reused once.

Quick destain
50% Methanol
7.5% glacial acetic acid

quick destain for no more than 5 minutes to remove precipitated stain.

5% methanol
7.5% glacial acetic acid

destain overnight with ~2 changes or until background is sufficiently low.

Good Luck!

Eric Hebert
Texas A&M University
Dept Medical Biochemistry and Genetics
Telephone (409) 845-6834 Fax 409-847-9481

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