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Rchqing's Content

There have been 12 items by Rchqing (Search limited from 02-April 19)


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#141846 Does triple labeled protein precipitate more easily?

Posted by Rchqing on 19 September 2012 - 05:13 PM in Protein Expression and Purification

I don't think addition of different isotopes will change the precipitability substantially. The colour is more likely to be from a buffer or something in the prep (detergents?) oxidizing.



Thank you! But only one of the buffer contains detergent (DPC), the other doesn't.



#141770 Does triple labeled protein precipitate more easily?

Posted by Rchqing on 19 September 2012 - 04:52 AM in Protein Expression and Purification

Hi, everyone!

I purified two different proteins these days. Both were labeled with 2H, 15N and 13C. I found they were light yellow when concentrated. However, the same unlabeled proteins I had purified previously were colorless, even though concentrated to a higher concentration. I don't know why and I am wondering whether triple labeled proteins are more likely to precipitate. Is there anyone can help with my questions? Thank you very much!



#141602 Method for protein concentration measurement

Posted by Rchqing on 16 September 2012 - 06:15 PM in Biochemistry



If you know the amino acid sequence then I would trust the Native scan. You could determine the concentration by denaturing your protein with some NaOH and using the extinction coefficient of your protein with the Tyrosines and Tryptophans at 260nm (if you have these amino acids).

Thank you very much! Could you give some suggestions about how much NaOH is needed?

We use a final NaOH concentration of 0.1N in the cuvette. Also, make sure the cuvette you're using is designed to be used in the UV range.


OK! Thank you very much!



#141519 Method for protein concentration measurement

Posted by Rchqing on 14 September 2012 - 09:39 PM in Biochemistry

If you know the amino acid sequence then I would trust the Native scan. You could determine the concentration by denaturing your protein with some NaOH and using the extinction coefficient of your protein with the Tyrosines and Tryptophans at 260nm (if you have these amino acids).

Thank you very much! Could you give some suggestions about how much NaOH is needed?



#141137 Relationship between OD600 and bacterial number

Posted by Rchqing on 11 September 2012 - 12:52 AM in Microbiology

Hi, everyone!
I have a question about OD600 and bacterial number. Is there a linear relationship exists between OD600 and bacteria concentration for a certain bacteria strain? For example, if the OD600 of my culture increases from OD600~0.6 to 1.2, will the bacteria number doubles as well?

Thank you! I am looking forward for your reply.



#140884 Which kind of ATP do you usually use in you experiment?

Posted by Rchqing on 06 September 2012 - 08:02 PM in Protein Expression and Purification

OK! I think I need to order ATP disodium salt.



#140883 Method for protein concentration measurement

Posted by Rchqing on 06 September 2012 - 07:59 PM in Biochemistry

Hi, everyone!

I purified a protein several days ago and measured the concentration of the sample yesterday using two different method. First, it was measured by BCA kit. the concentration is 7.6mg/ml. Then I measured UV280 of the sample and the concentration was calculated to be 14.3mg/ml(I know the Ext. coefficient). This is very strange! I don't know which method is more convincible. Which method do you usually use for protein concentration measurement?

I am looking forward to your replies. Thank you!



#140635 Which kind of ATP do you usually use in you experiment?

Posted by Rchqing on 04 September 2012 - 06:39 AM in Protein Expression and Purification

Hi, everyone!
I need to order ATP to test the activity of an ATPase. However, I cannot find pure ATP which is not complex with sodium or magnesium in the market. Most of ATP sold in the market is in their salt form. I don't know why. Maybe ATP salt is more stable than free ATP. I want to know which kind of ATP do you usually choose for enzyme activity assay----ATP disodium salt, ATP magnesium salt or free ATP complex with no metal ion? If I want to use free ATP, where can I order this kind of ATP?
Thank you very much!



#140283 How OD600 influence on IPTG induction and protein expression

Posted by Rchqing on 30 August 2012 - 03:28 AM in Protein Expression and Purification

In different phases bacteria have a different internal machinery determined by different expression levels of different essential proteins. In the log phase the internal machinery of the bacteria is set to produce lots of proteins and fast division since enough food is available. For proper expression you need to have enough cell mass to produce your protein and these bacteria need to have enough healthy transcription/translation machinery to be able to produce your protein. This is ideal in the fast growing stage.

However, when your protein is toxic to the cells (after induction at OD600 of 0.6, the cells stay at ~0.6 for many hours), you might want to consider induction at 1.2. Moreover, there are people who induce at 0.3 (beginning of the log phase).

Andreea

Thank you very much!



#139945 How OD600 influence on IPTG induction and protein expression

Posted by Rchqing on 25 August 2012 - 12:28 AM in Protein Expression and Purification

Why people always induce in log phase? Is it because bacteria's physiological status in this stage is most suitable for induction, or because there is enough nutrients for expression, or some other reasons?



#139942 How OD600 influence on IPTG induction and protein expression

Posted by Rchqing on 25 August 2012 - 12:00 AM in Protein Expression and Purification

Bacteria follows the Lag, Log, stationary and decline phase of growth in a limited amount of medium. If you have to take bacteria in log phase of growth so it express good. More overnight culture you take means you are running risk of taking O.D. for Stationary / Decline phase culture (overnight O.D.) than from log phase.

Posted Image

Do you mean the start OD of 0.4 is too large if I want to induce at OD=0.6? What is the start OD do you usually use for protein expression? Thank you!



#139937 How OD600 influence on IPTG induction and protein expression

Posted by Rchqing on 24 August 2012 - 09:02 PM in Protein Expression and Purification

I want to know how OD600 influence on IPTG induction and protein expression? Take for example. 100ml overnight culture is transfered to 1L fresh media and the start OD600 is 0.4. If it was induced when OD reaches 0.6, would it be OK? Thank you!




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