Fungal cell lysis

Share protocols and ask for sample preparation advice.
karthikskamath
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Fungal cell lysis

Postby karthikskamath » Mon Dec 10, 2012 10:44 pm

Hi Folks,

I am interested in extracting proteins by bursting the cells of a filamentous (dirty!) fungus. My aim is to get total cellular proteins into solution so that I can separate them over SDS-PAGE followed by trypsinization and LC-MS/MS. I have been trying few chemical and mechanical methods. Following are my takes on each method;

1) Sonication in presence of 8M urea, 2M thiourea and CHAPS followed by probe sonication (kept on ice between cycles, Roche protease inhibitor with 1mM EDTA used): Method adapted from paper for preparing samples for 2DE.
Outcome: No much of high molecular proteins seen on gel. Big blob of (Possibly) degraded proteins (10KDa-15KDa) seen. Non-uniform movement and streaking of proteins on gel due to chemical interference. Even after acetone precipitation!

2) Glass bead beating, PBS with 1% Sodium Deoxycholate/SDS : (kept on ice between cycles, Roche protease inhibitor with 1mM EDTA used)
Outcome: Very less proteins seen, no streaking effect, Big blob of degraded proteins (10KDa-15KDa) seen. Lane seems to have less number protein bands.

Question:
1) Is there any specific lysis buffer suggestions for fungal lysis (since fungal cell wall is rich with lipids, sugars etc)? Ideally a composition which can help in reduction of protein degradation,and help in getting most of the proteins in soluble state.
2) Can someone share related articles which guides through fungal protein extraction (NOT FOR 2-DE)?
3) Did someone in the group have problem by not using fungal specific protease inhibitor?
4) Tips/Tricks/Tactics??

Thanks in advance!
Karthik

lucky24
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Postby lucky24 » Tue Dec 18, 2012 1:14 am

Hi Karthik,
Here is the link to homogenization (general) of biological samples. Hope that it will be helpful to you.
http://opsdiagnostics.com/applications/OPSD_2012_Disruption_Guide_v1.1.pdf

Regards

trendsetter
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Postby trendsetter » Thu Dec 20, 2012 7:00 am

Hi Karthik,
The buffers you are using are very good. I would use a normal SDS buffer for 1D-PAGE that includes boiling of samples. This should work best. You can find a protocol here: http://www.nature.com/protocolexchange/protocols/455#/procedure
Good luck!

karthikskamath
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Posts: 53
Joined: Thu Dec 15, 2011 7:58 am

Postby karthikskamath » Thu Dec 20, 2012 7:34 pm

Hi TRENDSETTER! Many thanks for the response. I am following the boiling process. However my problem seems to be degradation. As mentioned in the previous post, I can see a big blob sitting below 15KDa. I just finished analyzing the digests of the blob on Q-exactive and that band gave maximum number of IDs and many of those proteins have higher Mol.wt. That clearly indicates that those proteins do not belong to region below 15KDa, rather they are degraded products. Any suggestions for reducing the chances of degradation? I already have protease inhibitor and 1mM EDTA in my lysis buffer.

@Lucky24: Thanks. I am going through the article.


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