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        SDS-PAGE


   SDS-PAGE

SDS-PAGE also allows an estimation of the purity of protein samples. SDS is an anionic detergent and isused to denature the proteins. The negative charges on SDS destroy most of the secondary and tertiary structure of proteins, and are strongly attracted toward the a node in an electric field. Because the charge-to-mass ratio is nearly the same among SDS-denatured proteins, the final separation of proteins is almost entirely dependent on the differences in relative Mw of polypeptides. Some caution has to be put on intrinsic strong negative or strong positively charged proteins because SDS may bind to them differently and their migration in the gel may not be at the expected Mw. In PAGE the relative migration distance of a protein (Rf) is negatively proportional to the log of its Mw. To be able to estimate the Mw  of proteins on the SDS-page, proteins of known Mw need to be run simultaneously on the gel. These proteins are called Protein Standards (MB-201-0200).   

 

Beside the protein molecular weight marker Rockland also offers a 10% SDS solution (MB-015), SDS-PAGE running buffer (MB-017) and sample loading buffer (MB-018). 

 

 

Protocol: 

 

  

1.   Decide which percentage of gel you need to separate your proteins.Eg.:

   

  

    • Use 4-8% gels to separate proteins 100 to 500 kDa in size.

 

    • Use 4-20% gels to separate proteins 10 to 200 kDa in size.   

  

     

 

  

2.   Place your gel in a clean plastic electrophoresis chamber andcorresponding gel holder.   

  

     

 

  

3.   Prepare 1X SDS-PAGE Running Buffer as follows:  

  

    • for 500 mL of 1X SDS-PAGE Running Buffer by adding 50 mL of 10X SDS-PAGE Running Buffer (MB-017) to 450 mL of diH20 (MB-009-1000).  

  

     

 

 

  

4.  Fill the inner portion between the gel(s) and the gel holder withthe appropriate 1X Running Buffer. Pour the remaining 1X Running Buffer into the outer chamber.   

  

  

 

   

  

5.  Sample Preparation:  

  

 

    • If using a pre-prepared lysate (already in sample buffer), thawlysate and transfer 25 μL of lysate to a clean pre-labeled microcentrifugetube. Add BME to a final concentration of 0.55M, i.e. add 1 μL stock BME per 25μL lysate. Mix well by pipetting. Label microcentrifuge tubes with sampledescription, volume and concentration of lysate.   

 

  

    • Any other protein samples: transfer to clean pre-labeledmicrocentrifuge tubes and mixed with an equal volume of 2XSample Buffer(MB-018) with 0.55M β-mercaptoethanol. Sample protein concentration shouldbe sufficiently high;  eg.: final proteinconcentrations from 1 μg to 500 μg depending on protein type and detectionmethod.   

  

 

    • Prepare molecular weight standards (MB-201-0200) for electrophoresis. For SDS-PAGE use eitheran unstained molecular weight standards or pre-stained molecular weight marker.For SDS-PAGE followed by western blotting, use pre-stained molecular weightmarkers.

  

 

  

    • Record lane #, sample description, sample concentration,loading volume, loading amount and addition of reducing agent for all samples.

   

  

     

  

6.  Place all microcentrifuge tubes containing samples for SDS-PAGEinto a heating block (set to 95°C) or water bath. Heat samples for 5 min.   

  

 

    

  

7.  After heating, centrifuge the aliquots for 3 min using amicrocentrifuge to pellet any debris.   

  

  

   

  

8.  Load all samples into gel lanes starting with the molecularweight standards. Sample loading volumes should be from 5 μL to 35 μL per lane(depending on gel). If protein concentrations are from 100 μg/mL to 500 μg/mL,then sample amounts will range from 0.5 μg to 17.5 μg per lane. Generally, 1.0μg is sufficient to visualize purified proteins and 10 μg is sufficient tovisualize proteins in lysates on a coomassie stained gel.   

  

    

 

  

9.  Cover the chamber and firmly connect both the anode and thecathode. Set the voltage on the electrophoresis power supply to a constant voltage of 150 V. Turn ON the power supply.   

  

   

  

CAUTION! Do not touch the electrophoresis unit while power is on. If buffer is leaking fromthe unit be certain to TURN POWER OFF before making contact with buffer. Usecare at all times.  

  

 

    

  

10.  Allow the gel to electrophorese for 45-90 min. Turn OFF thepower immediately after the dye front migrates out from the bottom of the gel.

 

 

 

  

11.  Disconnect the electrodes and remove the cover. Remove gel holder from the electrophoresis chamber. Carefully remove the gel from holder. Remove the gel from its plates and proceed with desired detection method.