4 replies to this topic

[cher]

Hi. I am new to protein expression and purification and have problems troubleshooting.

I am expressing Influenza A protein, specifically NS1 and MP1 using BL21-A1 E.Coli cells. During my SDS page analysis, i ran my resuspended pellet (induced and uninduced) along side with the soluble and insoluble fractions (both induced and uninduced) that i have separated via centrifugation after incubation with lysozyme for 30mins and boiling at 95 degrees for 10 mins. Followign this, Coomassie staining of the SDS page gels shows that there is indeed overexpression in the lanes of my induced resuspended pellet as compared to the uninduced resuspended pellet. However, no corresponding bands appeared in both my soluble and insoluble fraction lanes. I am confused as to how the protein could have disappeared when it should at least appear in either the soluble or insoluble fraction.

Please share with me your experiences. Thank you.

[protolder]

Hola, The easiest explanation is that lisozyme digests your overexpressed proteins. What about sonication to broke cells?, in this way you have one more control of expression. Good luck

[protolder]

Hola, The easiest explanation is that lisozyme digests your overexpressed proteins. What about sonication to broke cells?, in this way you have one more control of expression. Good luck

[cher]

protolder, on 28 September 2010 - 02:00 AM, said:

Hola, The easiest explanation is that lisozyme digests your overexpressed proteins. What about sonication to broke cells?, in this way you have one more control of expression. Good luck

Hi thanks for your reply. If it is true that lysozyme digest my overexpressed proteins, then i can't use it. Would sonication break the cells sufficiently if i don't add lysozyme? And would adding a protease inhibitor help?

[protolder]

Hola, Yes, sonication is the most commun method to broke bacteria.Freezing and thawing cycles is other possibility. You can add protease inhibitors like PMSF or EDTA (if your protein hasn´t 6HIS tag, because if it has, you can´t use EDTA). There are commercial protease inhibitors coktails. Good luck