user_mobilelogo

 In-Gel Digestions

In-gel digestions are a convenient way to identify what is in your band. Just excise the gel band by cutting out only the band of interest, as close to the band as possible, and avoiding the edges of the lane. Please also submit a band that is "blank" to use as a background control.

Your in-gel sample MUST be submitted with a gel image. On the submission form, please indicate the type of stain used. If you are using silver stain, use caution to not over-develop the gel, the protein may become inaccessible and the digest will not be efficient. Please see our sample check-list here.

Excision of protein bands from polyacrylamide gels:

Wash the gel slab with water and use a clean scalpel to excise the spot of interest from the gel.  Cut as close to the protein band as possible to reduce the amount of "background" gel.   Excise a gel piece of roughly the same size from a non-protein containing region of the gel for use as a control. [ Note: if the protein of interest appears to be contaminated with other proteins migrating nearby then excise the control blank below the band of interest to obtain a representative pattern of contamination].  Cut the excised piece into roughly 1 mm3 cubes and transfer them to a clean 1.5 mL or 0.5 mL microfuge tube.

Washing of gel pieces:

Wash the gel particles with water and water/methanol 1:1 (v/v)  (two times with, 15 minutes). Solvent volumes used in the washing steps should roughly equal 5 times the gel volume.

Remove all liquid and add enough acetonitrile to cover the gel particles.  After the gel pieces have shrunk (they become white and stick together) remove the acetonitrile. Store at -80C.

In-Solution Digest

If you would like to identify the most abundant proteins in your sample, and in-solution digest might be the way to go. Consider the complexity of you sample- The more complex your sample is, the less information we will get for the low abundance proteins. In the case of total cell lysates, the sample will require some type of fractionation before running on the mass spec.

In-Solution samples MUST be submitted with a representative gel image, so the staff is able to determine the quality, quantity and complexity of the sample. We also MUST be told which buffers are in your sample. If any detergent is present, we will choose the most appropriate method for removing it, including a pre-digestion TCA/acetone precipitation, a filter-aided-sample-preparation (FASP), or a post digest clean-up using detergent removal spin columns or HILIC tips. The use of these techniques is sample dependent, so please discuss these options with the staff if you are concerned about your sample.

 

Please be cautious of keratin throughout the handling process, and wear gloves at all times. Clearly label your tube with your NAME, DATE and the sample name. 

 

 

 

Location

The Johns Hopkins Mass Spectrometry and Proteomic Facility

725 N. Wolfe Street
Wood Basic Science Building, Room 504
Baltimore, MD 21205

Newsletter Signup

Sign up for all of our updates.
Please wait