Any suggestions regarding purifying my protein?

08 August 2013 1 8K Report

I am purifying my protein with GE his-trap 1ml column, I have started with different imidazole concentration. I have my final concentration wast with 110mM imidazole wash and elution with 250mM. I still have another protein that is present in first 1ml wash. My protein is 45kDa and other protein is about 40kDa. My eluted protein concentration is very low too.

Jan Piwowarski

Suggest this 40kDa protein could be degradation form. To verify it you need to do simple Western-blot with anti-His antibody. What is the composition of your lysis/binding buffer? Could be there is the problem…

Badges
Science method

More Muhammad Faheem's questions See All

Enzyme pH optimization protocol?

I am optimizing pH for my cellulase enzyme with substrate CMC and pNPG. Can anyone provide me with a good protocol for pH optimization?

09 October 2013 3,213 6 View

Protein purification and enzyme activity?

I am working with cellulases, I have expressed my cellulase enzyme in pET28a expression vector. The bacterial lysate has activity against cellulose substrate but after purification of protein with...

08 September 2013 2,331 9 View

His-tagged protein purification?

Could anyone suggest me a good protocol for purification of my his-tagged protein. I am eluting my N-terminus his-tagged protein with imidazole but after purification my protein has no more...

08 September 2013 3,311 5 View

His-tagged protein purification with GE Hi-trap 1ml column?

I am purifying my his-tagged protein with GE His-trap 1ml column. I started with equilibration of the column with 10mM and 20mM imidazole, and washing has been raised to 50mM, 100mM, and 110mM...

07 August 2013 2,632 9 View

I have to double digest my gene in a Plasmid, but the buffers are not compatible, what should I do?

31 December 2012 3,835 12 View

Can we classify HV and LV lines based on line length?

I have dataset which shows the length of power lines. I need to classify the lines based on the line length. Is there a rule to classify the High voltage (HV) and low voltage (LV) lines based on...

03 March 2021 4,116 4 View

Enhanced Yellow Fluorescent Protein (Aequorea victoria) DNA sequence?

I am on the lookout for the Enhanced Yellow Fluorescent Protein (Aequorea victoria) DNA sequence. Does anyone know where I can find it? Thank you in advance

03 March 2021 3,568 1 View

Is There Any Feasible Method To Test Fluorescent Compounds Other Than Fluorescence Spectrometers ??

Is There Any Feasible Method To Test The Efficiency Of Fluorescent Compounds Other Than UV Spectrometers ? Suggestions Would Be Appreciated !

02 March 2021 5,785 3 View

New Journal, "Integrative Psychological and Behavioral Science" -- do you not know, and have you not seen, this done before?

Question to you and THEM, the New Journal, "Integrative Psychological and Behavioral Science" -- do you not know, and have you not seen, this done before? There appears to be a core problem for...

02 March 2021 3,024 2 View

Does anyone know HRM channel in a 2 plex QIAGEN Rotor- Gene Q qPCR can be used as a separate channel?

I am going to have 3 different probes in my qPCR work that I am going to do. But I realized that the machine we have in the lab is a Rotor-Gene Q 2plex HRM Platform, saying it has green, yellow,...

01 March 2021 8,544 1 View

Why might my GFP tag be dissociating from my plasmid after transfection?

I have a set of stably transfected cell lines all transfected with plasmids containing GFP tags on the C terminus. During a western blot using anti-GFP antibody, one of my plasmids has dissociated...

01 March 2021 9,310 4 View

Precipitate after adding laemmli buffer to protein solution containing Potassium Acetate?

Hi, I am running a size exclusion chromatography experiment with a buffer containing Potassium Acetate as a salt. I analyse these fractions through SDS-PAGE. After boiling my SEC fractions in...

01 March 2021 2,622 3 View

What are the most relevant MSc proposal topics for Petroleum Production Engineering at the moment?

I am a Petroleum Production Engg MSc student at Robert Gordon University in Aberdeen and I am in the process of selecting a topic for my MSc Engineering Investigation proposal. I would very much...

01 March 2021 7,396 4 View

What is the most suitable server for predicting protein structures in bacteria?

I have used the i-Tasser several times, however it has been unavailable for several days. I tried the swiss-model, but the output was not very pleasant due to the model used. Is there any other...

28 February 2021 4,521 3 View

Do you need the CMV enhancer before the CMV promotor for a proper function of CMV on the gene that follows?

I'm a student and I have to produce a cell line with a knockin for the NRF2 gene with GFP. I have to put a promotor in front of the GFP because the gene will be too far away from the promotor of...

28 February 2021 7,127 2 View