Is My Protein Pure or Not? - Protein purification, SDS-Page (Feb/11/2011 )
Hi Everyone,
This is my second post in this section.
Attached herewith is the pic of my SDS-Page of protein purification that I've done.
The 8,9 and 10 column were the protein that I need to purify.
Based from the bands that appear, I would like to confirm whether it is pure or not. Any advice,suggestion and ideas are welcome.
Thank you.
-rpjkmust916-
that depends on the subunit structure of your protein and what you will do with the protein.
there appears to be some minor (possibly insignificant) high molecular weight contamination.
the band just below the protein of interest (i assume) is significant. is it a subunit? degraded protein of interest? will it interfere with the usage of the protein?
you can express purity as a percentage of the total if you evaluate the lanes.
you can also load a large amount of protein and see what else shows up.
-mdfenko-
Thanks mdfenko 
,
mdfenko on Fri Feb 11 20:34:27 2011 said:
that depends on the subunit structure of your protein and what you will do with the protein.
I need to purify the protein 95% pure at least.
mdfenko on Fri Feb 11 20:34:27 2011 said:
there appears to be some minor (possibly insignificant) high molecular weight contamination.
the band just below the protein of interest (i assume) is significant.
When you mentioned it, I noticed too, that it has high molecular weight contamination which is shown by the band below of the protein of interest (yes, the darker band at 43KDa)
mdfenko on Fri Feb 11 20:34:27 2011 said:
is it a subunit? degraded protein of interest? will it interfere with the usage of the protein?
Actually my knowledge in protein expression and purification is so little.
May I ask what do you mean by 'subunit, degraded protein of interest'?
I'm not sure whether the contaminate will interfere with the usage of the protein. It might cause an interference , I think. That's why my supervisor really angry with me when I showed him this gel. He also asked me why there were two bands.
mdfenko on Fri Feb 11 20:34:27 2011 said:
you can express purity as a percentage of the total if you evaluate the lanes.
you can also load a large amount of protein and see what else shows up.
Could you elaborate more about this, please. Do I have to measure the protein concentration before I load it to the gel? How I can evaluate the lanes?
I'm sorry for asking a lot of question.
Thank you for spending time to answer my questions.
-rpjkmust916-
rpjkmust916'>
is it a subunit? degraded protein of interest? will it interfere with the usage of the protein?
Actually my knowledge in protein expression and purification is so little.
May I ask what do you mean by 'subunit, degraded protein of interest'?
I'm not sure whether the contaminate will interfere with the usage of the protein. It might cause an interference , I think. That's why my supervisor really angry with me when I showed him this gel. He also asked me why there were two bands.
I think his point is that, your protein could possibly be pure, but if there are some degradation products those would show up as different bands in your gel because they are only fragments of the protein of interest. Another possibility could be formation of dimers, trimers, etc. that would form higher molecular weight bands.
-proteaMatt-
rpjkmust916 on Sat Feb 12 03:47:06 2011 said:
mdfenko on Fri Feb 11 20:34:27 2011 said:
is it a subunit? degraded protein of interest? will it interfere with the usage of the protein?
Actually my knowledge in protein expression and purification is so little.
May I ask what do you mean by 'subunit, degraded protein of interest'?
I'm not sure whether the contaminate will interfere with the usage of the protein. It might cause an interference , I think. That's why my supervisor really angry with me when I showed him this gel. He also asked me why there were two bands.
(your supervisor shouldn't get angry) many proteins are made up of multiple protein chains, called subunits. sometimes they are the same (eg- hemoglobin), sometimes they are different (eg-igg heavy and light chains). the two bands you see could be subunits of the protein. are they in the same ratio for each sample? do you know the native molecular weight of the protein? how do these bands compare? do they both stain in a western blot? if so, are you using polyclonal or monoclonal antibodies?
the lower band could be a fragment of your protein of interest.
mdfenko on Fri Feb 11 20:34:27 2011 said:
you can express purity as a percentage of the total if you evaluate the lanes.
you can also load a large amount of protein and see what else shows up.
Could you elaborate more about this, please. Do I have to measure the protein concentration before I load it to the gel? How I can evaluate the lanes?
you should always have an idea of how much protein you load in each lane but that is not really necessary to determine percent purity.
you can evaluate the lanes by either scanning them or by imaging them (take a picture) and determine the relative amount of each band in each lane (you can use a program like imagej to evaluate the image).
-mdfenko-
Thanks proteaMatt and mdfenko,
Now my knowledge in protein is increasing, may I ask more
mdfenko on Tue Feb 15 21:11:57 2011 said:
the two bands you see could be subunits of the protein. are they in the same ratio for each sample? do you know the native molecular weight of the protein? how do these bands compare? do they both stain in a western blot? if so, are you using polyclonal or monoclonal antibodies?
the lower band could be a fragment of your protein of interest.
May I know, what do you mean 'in the same ratio for each sample'?
I have not learn to do the western blot yet
Is it possible to identify it as a fragment if I run western blot?
About the native molecular weight of the protein, let me give the details
It is a nuclear receptor protein with 280-486 aa, cloned into PET 32a Rosetta.
My senior told me the molecular weight of this protein is 43KDa.
The calculation she showed me was like this,
- 206 aa x 110 Da = 22660 so ~23KDa
- 23KDa + 20 = 43KDa
Actually, I don't understand
But I'm afraid to ask for more because we do have language barrier and she seems like not in good condition lately.
mdfenko on Tue Feb 15 21:11:57 2011 said:
you should always have an idea of how much protein you load in each lane but that is not really necessary to determine percent purity.
you can evaluate the lanes by either scanning them or by imaging them (take a picture) and determine the relative amount of each band in each lane (you can use a program like imagej to evaluate the image).
Thanks for the explanation.
There is no scanning machine for the gel in the lab. I only scanned the gel with scanner that attached with the computer.
Thank you again and again.....
-rpjkmust916-
rpjkmust916 on Thu Feb 17 02:22:57 2011 said:
Thanks proteaMatt and mdfenko,
Now my knowledge in protein is increasing, may I ask more
mdfenko on Tue Feb 15 21:11:57 2011 said:
the two bands you see could be subunits of the protein. are they in the same ratio for each sample? do you know the native molecular weight of the protein? how do these bands compare? do they both stain in a western blot? if so, are you using polyclonal or monoclonal antibodies?
the lower band could be a fragment of your protein of interest.
May I know, what do you mean 'in the same ratio for each sample'?
I have not learn to do the western blot yet
Is it possible to identify it as a fragment if I run western blot?
About the native molecular weight of the protein, let me give the details
It is a nuclear receptor protein with 280-486 aa, cloned into PET 32a Rosetta.
My senior told me the molecular weight of this protein is 43KDa.
The calculation she showed me was like this,
- 206 aa x 110 Da = 22660 so ~23KDa
- 23KDa + 20 = 43KDa
Actually, I don't understand
But I'm afraid to ask for more because we do have language barrier and she seems like not in good condition lately.
mdfenko on Tue Feb 15 21:11:57 2011 said:
you should always have an idea of how much protein you load in each lane but that is not really necessary to determine percent purity.
you can evaluate the lanes by either scanning them or by imaging them (take a picture) and determine the relative amount of each band in each lane (you can use a program like imagej to evaluate the image).
Thanks for the explanation.
There is no scanning machine for the gel in the lab. I only scanned the gel with scanner that attached with the computer.
Thank you again and again.....
On referring to your pet32a vector, Is your protein Tagged (Trx, S, His)?
CAn you explain into detail about your work flow? Do you pre-treat your protein
Based on your calculation from your senior, I assume that you did not pre-treat your protein with thrombin/enterokinase before purify your protein through the column.
The Trx tag, s tag and His tag together forms roughly 158aa before your protein.
So 158 x 110 = 17380 ~17kDa
You probably use HindIII or SalI RE to cut the vector for your cloning which adds around ~18aa (~2kDa)(rough calculation) so the total would be around 20kDa (17+2=19, ~20) contributed from your vector.
Your cloned in protein was 206aa, which you suggested to be ~23kDa. So in total, you should get 23 + 20 =~ 43kDa protein.
P/S: Slightly off track from your topic, I really not sure whether is it a good idea to have so many "tag" before your protein, as your "tag" was almost the same size with your protein, and wouldn't it affect your protein functions?
-adrian kohsf-
Thanks Adrian ,
adrian kohsf on Thu Feb 17 05:29:45 2011 said:
On referring to your pet32a vector, Is your protein Tagged (Trx, S, His)?
CAn you explain into detail about your work flow? Do you pre-treat your protein
It was His-Tag.
About my work flow, do you mean by the expression process?
I've never done the cloning part.
I only do the culture by starting with small culture (culture 1 colony in 10 ml LB with antibiotics) and incubate it for overnight at 37
0C.
After that transfer it to 1L of LB (with antibiotics) and incubate again untill it reach the OD
600 = 0.6-1.0.
Then, I add 0.1mM IPTG and incubate at 18
o C for 20hrs.
Next, I harvest the cell and discard the supernatant.
Lysis the pellet with sonicator and filter it (I did not treat the lysate with any protease inhibitor).
Centrifuge and pool all the supernatant (now I know it also known as 'soluble protein'
).
And finally, run the purification with the supernatant.
I'm using HiTrap Chelating HP (5ml)affinity column.
The buffers that I used for the column as listed below:
Running/Lysis buffer: 50mM Tris, 5mM 2-Me
Elution buffer : 50mM Tris, 5mM 2-Me, 500mM Imidazole
adrian kohsf on Thu Feb 17 05:29:45 2011 said:
P/S: Slightly off track from your topic, I really not sure whether is it a good idea to have so many "tag" before your protein, as your "tag" was almost the same size with your protein, and wouldn't it affect your protein functions?
Actually, I'm not fully understand. Could you explain,please?
Anyway, thank you very much Adrian
P/S: It looks like more explanation given, I ask more and more. Apologize for any inconvenience
I'm a newbie in molecular biology
.
-rpjkmust916-
rpjkmust916 on Thu Feb 17 06:22:41 2011 said:
Thanks Adrian
,
adrian kohsf on Thu Feb 17 05:29:45 2011 said:
On referring to your pet32a vector, Is your protein Tagged (Trx, S, His)?
CAn you explain into detail about your work flow? Do you pre-treat your protein
It was His-Tag.
About my work flow, do you mean by the expression process?
I've never done the cloning part.
I only do the culture by starting with small culture (culture 1 colony in 10 ml LB with antibiotics) and incubate it for overnight at 37
0C.
After that transfer it to 1L of LB (with antibiotics) and incubate again untill it reach the OD
600 = 0.6-1.0.
Then, I add 0.1mM IPTG and incubate at 18
o C for 20hrs.
Next, I harvest the cell and discard the supernatant.
Lysis the pellet with sonicator and filter it (I did not treat the lysate with any protease inhibitor).
Centrifuge and pool all the supernatant (now I know it also known as 'soluble protein'
).
And finally, run the purification with the supernatant.
I'm using HiTrap Chelating HP (5ml)affinity column.
The buffers that I used for the column as listed below:
Running/Lysis buffer: 50mM Tris, 5mM 2-Me
Elution buffer : 50mM Tris, 5mM 2-Me, 500mM Imidazole
adrian kohsf on Thu Feb 17 05:29:45 2011 said:
P/S: Slightly off track from your topic, I really not sure whether is it a good idea to have so many "tag" before your protein, as your "tag" was almost the same size with your protein, and wouldn't it affect your protein functions?
Actually, I'm not fully understand. Could you explain,please?
Anyway, thank you very much Adrian
P/S: It looks like more explanation given, I ask more and more. Apologize for any inconvenience
I'm a newbie in molecular biology
.
Hmn, exactly what I want to ask about your cloning strategy (did you removed any of the "tag" or modify them), but then you said you did not do the cloning work...
Forget about what I had asked in previous since you might not know exactly the construct.... lets see, what is your purpose of using this recombinant protein? If you do not need a native protein, you can always excise the band and do electro elution to get a pure band. How is that?
-adrian kohsf-
Thanks for the reply Adrian,
My senior told me that she did not remove the His-tag. I have to purify the protein for my colleague at concentration 1mg/ml. I'm not really sure of her works/experiment.
According to my senior, the his-tag should not be a problem for the experiment.
If I excise the band and electro elution it, how about the concentration?
The main concern here is to get 95% purity of the protein at mass 1mg/ml.....*sigh*
Anyway, thanks Adrian for the suggestion. I've gained a lot about protein purification here
Still need to read a lot.
Just a silly question...is there any book like "Protein Purification for Dummies" like me 
Any suggestions, advices, opinions are mostly welcome
Thank you.....Kamsahamnida....Ariatou gozaimasu...last but not least....Terima Kasih!!!!
-rpjkmust916-
