Loading control or marker (reference marker)? If you are looking for something that remains in a 6% gel after the run is complete and whose levels are constant, I would recommend HAT p300 (for WB analysis). It runs at 300 kDa. Depending on the cell line, though, its expression may be different. But its levels stay constant within the same cell line under many different stimuli/treatments. Other possible candidates: Brg1 and Brm (key subunits of SWI/SNF complexes, run at ~150 kDa). In case you are looking for the marker within that range, I would recommend HiMark pre-stained (Life Technologies).

Dear Melissa,

I suggest to use Tubulin (50-55 kDa ) as a loading control.

Regards

Mellisa,

I understand your question but what I don’t understand why you really need a loading control to be probed around 300 kDa? As loading controls could be any protein whose expression ideally should remain constant irrespective of the experimental treatment conditions. Since you are running 6% SDS PAGE (No gradient) for your experiment, I guess you are running too far your SDS PAGE to let the high molecule protein enter. Usually for 6% SDS PAGE ran on Bio-Rad Protean III or Tetra, proteins of molecular weight ~205kDa enter without any problem which could be successfully transferred onto NCP/PVDF for western analysis.

If you have access to any other electrophoresis apparatus that allows you to have a run for longer duration without eluting any protein through the gel you will definitely be able to see the mid mol wt range loading control proteins ( may be beta actin, 43 kDa or Tubulin , 55 kDa). For membrane proteins Na,K-ATPase alpha 1 subunit is also frequently probed as a loading control.

Without knowing the location for the proteins that you are interested in and the protocol for your sample preparation it may not be wise to comment anything about choosing proper loading controls as proteins are specifically and differentially expressed in cellular sub-organelles and compartments (nuclear, cytosolic, ER, plasma membrane etc.) depending upon their designated function(s)/role(s). Still if you really want to look for other proteins which run ~300kDa, you can try probing for fibronectin (assuming you don’t have any TGFb1 stimulator in your experimental condition as TGFb1 stimulators are the known modulator of fibronectin expression and you are working with whole cell lysate). You can try Abcam (ab6328) for this purpose.

Hope that helps!!!

Just an additional comment:

I am not sure whether 6% SDS PAGE ( you just mentioned the % of the gel, no information provided for the composition) is the right gel that you selected for resolving proteins for such a high molecular weight (350-500kDa). My understanding is that NuPAGE® Novex Tris-Acetate Gels (gradient 3-8%) would be the proper gel to resolve your protein of interest. You can consider running high molecular wt standards as well (http://www.lifetechnologies.com/order/catalog/product/LP0001) to identify the exact molecular weight of your protein under denaturing condition.

For high molecular weight proteins I was using Vinculin as a loading control (as an example: 66305-1-Ig CloneNo.: 2B5A7 Vinculin mouse monoclonal).