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Keck Home Page > Protein Chemistry > Sample Preparation Sample Preparation for In Situ Enzymatic Digestion in SDS Page Gels General Instructions Laemmli and most other types of 1D and 2D gels, ranging from 7.5% to 17.5%, may be used. In general, we recommend using the higher percentage gels (e.g., 15% for proteins less than 30 kD) to minimize loss of sample during staining/destaining and our washing of the submitted gel bands. Samples should be submitted in the smallest gel volume possible. Hence, if possible, use 0.5 mm to 0.75 mm thick gels (rather than 1.0 to 1.5 mm) and keep the protein sample in a single gel lane such that the final density of the protein in the stained gel will at least 0.05 µg/mm3. If there are technical problems that prevent loading the sample in a single gel lane, we suggest you email a short description of the problem to the Protein Chemistry Section who may be able to provide assistance. To aid in quantifying the sample, we urge that all investigators load several concentrations of a known mixture of several standard proteins in non-adjacent lanes of the same gel. Since Coomassie Blue staining intensity varies by at least two-fold (depending upon the protein) it is important that more than one standard protein be run on the same gel as the sample and that an average staining intensity be used to estimate the amount of protein in the sample. Since this estimate may well determine the procedures that may be taken to either identify the protein or to obtain internal Edman sequences, it is extremely important that the estimate of the amount of protein submitted be as accurate as reasonably possible. We recommend that gels be stained with Coomassie Blue (either R250 or G250). In the case of G250 (colloidal) Coomassie Blue, the sensitivity of staining appears to be about 25 ng ( i.e., 0.3 pmol) transferrin in a 0.5 mm thick gel. Currently, we do not recommend the use of silver staining. If the protein of interest is localized via some means other than staining (e.g. comparison to a parallel, stained gel), it is important that the gel be well washed to remove the very high concentrations of SDS, Tris and other transfer buffers. In this case we suggest bringing the gel through the following Coomassie Blue staining protocol without actually adding Coomassie Blue to the acetic acid/methanol solvent. Coomassie Blue Staining Stain the gel with 0.1% (or less) Coomassie Blue R250 in 10% acetic acid, 50% methanol, and 40% H2O for the minimum time (typically less than one hour) necessary to visualize the bands of interest. If the protein can be localized by staining a guide strip or be visualized with a lower Coomassie Blue concentration, every effort should be made to limit the amount of Coomassie Blue that is used. However, regardless of the [Coomassie Blue], the gel should be exposed to 10% acetic acid, 50% methanol for a total (stain plus destain) period of at least 3 hours (with shaking and at least three solvent changes) to ensure adequate removal of SDS. Destain the gel by soaking for at least 2 hours in 10% acetic acid, 50% methanol, and 40% H2O with at least two changes of this solvent. If the gel still has a Coomassie Blue background then continue destaining until the background is nearly clear. In the case of colloidal Coomassie Blue G250, we have followed the vendor's (e.g., Sigma B 2025) recommendations for staining/destaining. Silver Staining If the protein of interest can be visualized only by the use of silver staining, we recommend using the protocol described briefly in Shevchenko et al ('96). After washing the gel with 5% and then briefly with 1% acetic acid, the spots or bands of interest should be excised and placed (without any additional liquid) into Eppendorf tubes and then frozen and shipped as described below. Although we have not independently tested the method described by Gharahdaghi et al ('99) for removing silver ions, the data presented in this paper suggests this approach results in increased sensitivity. Preparation of Samples for Manual MALDI-MS Protein Identification by Peptide Mass Database Searching Excise the band from the gel in such a manner as to avoid removing excess gel that does not contain any protein. Place the excised band in an Eppendorf tube (that does not contain a rubber O-ring) and freeze. Do not dry it or leave the gel band in destain buffer or in any other liquid. Excise a similar size piece of "blank" gel (that does not contain any protein) and put it in a separate Eppendorf tube so that this blank section of gel can be used as a control to identify HPLC artifact peaks. Preparation of Samples for Robotic Digestion Excise the stained bands and/or spots from the gel in such a manner as to avoid removing excess gel that does not contain any protein. Place each band (which must be less than 2 mm x 10 mm x 1.5 mm in size) or spot into an individual well (beginning with the A1, B1, C1, etc wells) in a Corning Costar #3363 V-bottom polypropylene 96 well plate. We suggest that you include at least one spot or band which derives from an area of your gel which should not contain protein and which can serve as a control. The minimum number of samples (including the control) that may be submitted is 8, which must be in wells A1 through H1. Please note that your samples will be identified by the Keck Laboratory only by the plate name you assign and then by their individual well designation. No liquid should be present in these wells. After covering the plates with a Corning #3080 Storage Mat III, they should be securely wrapped in Parafilm to prevent the lid from coming off and then stored at -20 degrees C prior to shipping to the Keck Laboratory. Shipment of Protein Identification Samples to the Keck Laboratory We recommend samples be sent (either with or without dry ice) via Federal Express (note that other overnight services will leave your package on the loading platform while Federal Express will deliver it directly to our laboratory). All samples must be accompanied by a completely filled out sample submission form that should be enclosed with the sample and packages should be addressed to: Ms. Mary LoPresti W.M. Keck Facility Yale University Room G001 300 George Street New Haven, CT 06511 Please be sure to include charging instructions on the order form as we cannot begin work on your samples until we have received valid charging instructions. If you do not receive a FAX (indicating our receipt of your sample) by 4 PM (EST) on the day your sample should arrive at the Keck Facility, please send an email to Mary LoPresti to confirm it's receipt.