We are interested in detecting aggrecan (250 KDa) by WB. No luck so far, are there any recommendations, such as running buffer, sample loading buffer? Thank you in advance!
Detecting aggrecan in Western blotting of mouse brain homogenates can be challenging due to its large molecular weight (~250 kDa). However, with careful optimization of experimental conditions and antibody selection, it is feasible. Here are key points:
By meticulously optimizing each step and addressing specific challenges associated with detecting large proteins like aggrecan, successful detection of the 250 kDa band in mouse brain homogenates can be achieved.
Dear Kais, I truly appreciate your pointed out the key points. My concern is that we are detecting lower molecular weight bands about ~75 kDa in WT mouse brain lysates. A friend mentioned the importance of glycosylation state... Thank you
Dear Claudia,
Along the lines of what Kaisers suggested, some key considerations include:
Sample: Do the samples need to be prepared on a specific way to release the protein monomers? Do boiling affects aggrecan's multimerization?
Gel: Choose a pretty low percentage gel to allow the migration of the large size protein. It might be necessary let let SDS-PAGE to run a bit longer than usual. Are you able to see the high-size molecular weight markers after transfer to the membrane?
Please take a look at:
Arthritis Res Ther. 2009; 11(3): R92. PMID: 19545413
J Biol Chem 281(26):17789-800. doi: 10.1074/jbc.M600544200.
Dear Marco, I truly appreciate your suggestions and insights! The information you shared here is super helpful. Thank YOU!
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