5 replies to this topic

[deepbluedna]

hi, I have my protein purified with his-tag successfully, and trying to cut the his-tag off with thrombin. The cleavage looks ok from the sds gel. (One thing I want to mention here is from the sds gel I can only see a thick band of my protein but no thrombin there at all.) So the only thing I need to do is to get rid of thrombin. I was trying to use benzamidine beads to wash the thrombin off but my protein stuck to the benzamidine beads too. My protein didn't come off till  I  increased the NaCl concentration till 1M.
So my question is since my protein is highly concentrated from the sds-gel relative to the thrombin in the solution (like 240:1), do you think I still need to use benzamidine to try getting rid of thrombin? in which case I will lose a lot of my protein. Thanks in advance!

[klinmed]

deepbluedna, on Jan 2 2010, 02:27 AM, said:

hi, I have my protein purified with his-tag successfully, and trying to cut the his-tag off with thrombin. The cleavage looks ok from the sds gel. (One thing I want to mention here is from the sds gel I can only see a thick band of my protein but no thrombin there at all.) So the only thing I need to do is to get rid of thrombin. I was trying to use benzamidine beads to wash the thrombin off but my protein stuck to the benzamidine beads too. My protein didn't come off till  I  increased the NaCl concentration till 1M.
So my question is since my protein is highly concentrated from the sds-gel relative to the thrombin in the solution (like 240:1), do you think I still need to use benzamidine to try getting rid of thrombin? in which case I will lose a lot of my protein. Thanks in advance!

The need to remove thrombin (and other minor contaminants) following cleavage depends on the downstream use of your recombinant protein. If it is to be used for immunizations, enzyme studies etc thrombin removal would probably not be needed. However, for crystallization further purification would be mandatory.

If you choose not to remove the thrombin you WILL need to inactivate it in order to prevent over-digestion. Addition of an irreversible serine protease inhibitor such as PMSF (1 mM final) following digestion should work fine.

Hope this helps

[deepbluedna]

klinmed, on Jan 3 2010, 09:18 AM, said:

deepbluedna, on Jan 2 2010, 02:27 AM, said:

hi, I have my protein purified with his-tag successfully, and trying to cut the his-tag off with thrombin. The cleavage looks ok from the sds gel. (One thing I want to mention here is from the sds gel I can only see a thick band of my protein but no thrombin there at all.) So the only thing I need to do is to get rid of thrombin. I was trying to use benzamidine beads to wash the thrombin off but my protein stuck to the benzamidine beads too. My protein didn't come off till  I  increased the NaCl concentration till 1M.
So my question is since my protein is highly concentrated from the sds-gel relative to the thrombin in the solution (like 240:1), do you think I still need to use benzamidine to try getting rid of thrombin? in which case I will lose a lot of my protein. Thanks in advance!

The need to remove thrombin (and other minor contaminants) following cleavage depends on the downstream use of your recombinant protein. If it is to be used for immunizations, enzyme studies etc thrombin removal would probably not be needed. However, for crystallization further purification would be mandatory.

If you choose not to remove the thrombin you WILL need to inactivate it in order to prevent over-digestion. Addition of an irreversible serine protease inhibitor such as PMSF (1 mM final) following digestion should work fine.

Hope this helps

thanks:)

[deepbluedna]

klinmed, on Jan 3 2010, 10:18 AM, said:

deepbluedna, on Jan 2 2010, 02:27 AM, said:

hi, I have my protein purified with his-tag successfully, and trying to cut the his-tag off with thrombin. The cleavage looks ok from the sds gel. (One thing I want to mention here is from the sds gel I can only see a thick band of my protein but no thrombin there at all.) So the only thing I need to do is to get rid of thrombin. I was trying to use benzamidine beads to wash the thrombin off but my protein stuck to the benzamidine beads too. My protein didn't come off till  I  increased the NaCl concentration till 1M.
So my question is since my protein is highly concentrated from the sds-gel relative to the thrombin in the solution (like 240:1), do you think I still need to use benzamidine to try getting rid of thrombin? in which case I will lose a lot of my protein. Thanks in advance!

The need to remove thrombin (and other minor contaminants) following cleavage depends on the downstream use of your recombinant protein. If it is to be used for immunizations, enzyme studies etc thrombin removal would probably not be needed. However, for crystallization further purification would be mandatory.

If you choose not to remove the thrombin you WILL need to inactivate it in order to prevent over-digestion. Addition of an irreversible serine protease inhibitor such as PMSF (1 mM final) following digestion should work fine.

Hope this helps

hi again,

what if i decide to get rid of thrombin? cause the protein is used for crystallization. Is there any other way to get rid of it?

[klinmed]

deepbluedna, on Jan 6 2010, 09:35 PM, said:

klinmed, on Jan 3 2010, 10:18 AM, said:

deepbluedna, on Jan 2 2010, 02:27 AM, said:

hi, I have my protein purified with his-tag successfully, and trying to cut the his-tag off with thrombin. The cleavage looks ok from the sds gel. (One thing I want to mention here is from the sds gel I can only see a thick band of my protein but no thrombin there at all.) So the only thing I need to do is to get rid of thrombin. I was trying to use benzamidine beads to wash the thrombin off but my protein stuck to the benzamidine beads too. My protein didn't come off till  I  increased the NaCl concentration till 1M.
So my question is since my protein is highly concentrated from the sds-gel relative to the thrombin in the solution (like 240:1), do you think I still need to use benzamidine to try getting rid of thrombin? in which case I will lose a lot of my protein. Thanks in advance!

The need to remove thrombin (and other minor contaminants) following cleavage depends on the downstream use of your recombinant protein. If it is to be used for immunizations, enzyme studies etc thrombin removal would probably not be needed. However, for crystallization further purification would be mandatory.

If you choose not to remove the thrombin you WILL need to inactivate it in order to prevent over-digestion. Addition of an irreversible serine protease inhibitor such as PMSF (1 mM final) following digestion should work fine.

Hope this helps

hi again,

what if i decide to get rid of thrombin? cause the protein is used for crystallization. Is there any other way to get rid of it?

A protein purified using a single IMAC step (eg via a his-tag) is usually not pure enough for crystallization (on an over-loaded gel you probably see multiple minor contaminants). You will need to perform at least one additional (and complementary) step. Ion exchange would probably be a good choice. By choosing the appropriate exchanger (anion/cation) and binding/elution conditions the majority of contaminants (including thrombin) will be removed.
If the Mr of your protein is significantly different from that of thrombin, gel filtration may be appropriate.

Hope this helps.

[deepbluedna]

klinmed, on Jan 6 2010, 02:23 PM, said:

deepbluedna, on Jan 6 2010, 09:35 PM, said:

klinmed, on Jan 3 2010, 10:18 AM, said:

deepbluedna, on Jan 2 2010, 02:27 AM, said:

hi, I have my protein purified with his-tag successfully, and trying to cut the his-tag off with thrombin. The cleavage looks ok from the sds gel. (One thing I want to mention here is from the sds gel I can only see a thick band of my protein but no thrombin there at all.) So the only thing I need to do is to get rid of thrombin. I was trying to use benzamidine beads to wash the thrombin off but my protein stuck to the benzamidine beads too. My protein didn't come off till  I  increased the NaCl concentration till 1M.
So my question is since my protein is highly concentrated from the sds-gel relative to the thrombin in the solution (like 240:1), do you think I still need to use benzamidine to try getting rid of thrombin? in which case I will lose a lot of my protein. Thanks in advance!

The need to remove thrombin (and other minor contaminants) following cleavage depends on the downstream use of your recombinant protein. If it is to be used for immunizations, enzyme studies etc thrombin removal would probably not be needed. However, for crystallization further purification would be mandatory.

If you choose not to remove the thrombin you WILL need to inactivate it in order to prevent over-digestion. Addition of an irreversible serine protease inhibitor such as PMSF (1 mM final) following digestion should work fine.

Hope this helps

hi again,

what if i decide to get rid of thrombin? cause the protein is used for crystallization. Is there any other way to get rid of it?

A protein purified using a single IMAC step (eg via a his-tag) is usually not pure enough for crystallization (on an over-loaded gel you probably see multiple minor contaminants). You will need to perform at least one additional (and complementary) step. Ion exchange would probably be a good choice. By choosing the appropriate exchanger (anion/cation) and binding/elution conditions the majority of contaminants (including thrombin) will be removed.
If the Mr of your protein is significantly different from that of thrombin, gel filtration may be appropriate.

Hope this helps.

thanks:) I think I am going to try the ion exchange method:) ill update it