Protocols

General Protein Isolation and IP Approaches:

Protein Extraction Reagents Pierce; MPER (cells), TPER (tissue) - manufacturer manuals

Cell Surface Protein Isolation (biotin tag pull down kit/ and a cell surface biotin web link)

Single Proteins/ Protein Complexes (Chatham Lab protocol)

Mitochondrial Protein Complexes (Hutson Lab protocol)

Protein/ Peptide Clean up and Quantification on a 96 well plate reader:

Protein and Peptide clean up (Microm CapTraps manual/ general protocol reverse phase)

Proteins EZQuant (fluorescence) - manufacturer manual

Proteins BCA (absorbance) - lab version

Peptides CBQCA (fluorescence) - manufacturer manual

Preferred Gel Staining Procedures:

Colloidal Coomassie (1D PAGE scanned on IR Odyssey scanner) - manufacturer

Sypro Ruby (2D PAGE scanned on Typhoon) - manufacturer

*note that staining with CC after SR improves visibility of 2D gels for manual spot picking.

Enzymatic Digestion Procedures:

(when there is enough sample, check the quality by running a portion 5-10 µg of the predigest on a 1D gel, check the efficiency of in-solution post digestions by running a portion 5-10 µg on a 1D gel, but ALWAYS QC all in-gel and in-solution digestions with a 100 ng of BSA protein standard run the same way side by side)

General In-Solution Digestion

Cell Lysate Prepration/ In-Solution Digestion

Tissue Lysate Preparation/ In-Solution Digestion

Common In-Gel Digestion

Common Protein Stds for LC/MS QC:

Pierce (BSA in PBS), 10-2mg/ml vials of 1 ml each. We digest directly as shown above for the in-solution procedures to use as a basic standard for LC/MS and MALDI, and also as conditioning media for our new columns. We also use this standard for all protein quants and as a standard to run on a gels along with test samples to monitor efficiency of digestion, staining, and overall runs.

Invitrogen (MARK-12) ~1mg of 12 different unmarked proteins in SDS solution; prep for LC/MS by standard in-solution digestion above following standard ice cold acetone precipitation 6.5:1.

Sigma (UPS-1) ~5pmole each of 48 different proteins; prep for LC/MS directly with any common in-solution digestion.

Sigma (UPS-2) ~10.6 mcg of 48 proteins diluted up to 5 orders in magnitude; prep for LC/MS directly with any common in-solution digestion.


Common Approach for MS workup (download general scheme)

Purified proteins from 2D PAGE or IP following 1D PAGE; MALDI-Tof/Tof

Complex proteins (i.e. 3-5 or more); LC-ESI-LTQXL


Reagent Catalog (Mobley Lab) Information (download order information)