{"id":1552,"date":"2020-04-02T13:54:52","date_gmt":"2020-04-02T12:54:52","guid":{"rendered":"https:\/\/sites.manchester.ac.uk\/bioms-community\/?p=1552"},"modified":"2020-04-02T17:01:03","modified_gmt":"2020-04-02T16:01:03","slug":"lab-gel-tops","status":"publish","type":"post","link":"https:\/\/sites.manchester.ac.uk\/bioms-community\/2020\/04\/02\/lab-gel-tops\/","title":{"rendered":"Lab methodolgies: Gel-tops"},"content":{"rendered":"

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Gel-top analysis is a method of preparing samples for in-gel digestion. The aim is to trap your entire sample in a small section of polyacrylamide gel for analysis as a single sample. We do not have a fixed protocol for preparing samples for ‘gel-top’ analysis but please follow the following guidelines:<\/span><\/p>\n

(i) It is important to keep the volume of gel in a digestion at a minimum as it reduces the digestion efficiency. We recommend a target of 5mm of gel up to a maximum of 10mm. This process should only take a couple of minutes at 200V but if in doubt use pre-stained markers and track the position of the lowest marker. Multiple loads are possible but this should be done with caution as it is hard to control the distance in the gel doing this, consider gels with higher volume wells such as Bolt gels<\/a> if you need to load more sample.\u00a0<\/p>\n

(ii) You should use a single concentration gel (e.g. a 10 or 12% gel) and never a gradient gel. This is because the low concentration at he top of a gradient gel is difficult to work with and many proteins will diffuse from this gel during processing.\u00a0<\/p>\n

(iii) Minimise contamination by only using clean containers for the gel that have not been used for any other purposes. We highly recommend using the square petri dishes that are available for stores for all gel handling. Keep the lid on as much as possible as much keratin contamination comes from dust in the atmosphere. Also be careful of any surfaces that the gel comes in contact with such as flat bed scanners as these may have fingerprints on them that will transfer on to the gel on contact producing further keratin contamination. If you need to image the gel then take an image from above whilst the gel remains in the clean container.<\/p>\n

(iv) Stain the gel in a quick coomassie stain such as Instant Blue. All the proteins in the sample will be piled on top of each other and so there should be no issue with sensitivity. Staining the gel allows us to know where your proteins are and where to cut.<\/p>\n

(v) Run your gel the day before you are booked in to use the in-gel digestion service and leave it overnight in the fridge in high quality water. This is to allow contaminants to defuse from the gel, improving the quality of your results. If your sample contains high concentration of detergents such as triton then allow more time for washing the gel and change the water at least three times. This is becuse these detergents form large micelles that only defuse slowly from the gel and even a small contamination can effect the results.<\/p>\n

Tags: #lab, gel-tops, PAGE<\/p>\n

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