Protein Expression and Purification Troubleshooting Forum

Insoluble Protein purification

Thu, 02 Feb 2012 21:25:18 +0000

Hi

First que - what is the difference between insoluble protein and inclusion bodies?

Second ques - I am trying to purify some insoluble Protein having the His-tag. My protein size is 30 kDA. I have tried the small scale 50 ml expression test where I soaked/resuspended my cell pellet in the Lysis buffer (50mM NaH2PO4, 300mM NaCl, 10mM Imidazole, 5% of Sarkosyl, pH-8.0). I diluted my Supernatant from 5% to 0.3% sarkosyl in the final volume and loaded to the Ni-NTA Spin column Qiagen (b'coz of the compatibility issue with Ni Column). The small scale test looks promising on gel and I was able to solubilize 50% of my insoluble protein. I can see a clear band in supernatant which has 50% intensity if I consider the Crude Cell lysate as 100%. However, not able to purify in the final stage. (It might not bind to the column) I am planning to repeat the same thing with big scale expression of 6 litre.

Does anyone provide me any detailed protocol for the His-tag protein expression and purification along with Srakosyl in buffer (Native condition)? I am not sure how much Supernatant should I load to the big Ni column (HiTrap Chelating 5 ml× 2 GE Health Care). I might have to dilute it from 5% to 1% as the big column is compatibile with 1% sarkosyl. I am not sure how much protease inhibitor and DNAse need to be added to my Cell pellet before I start purification ?

Correct protein expression in pichia??

Wed, 01 Feb 2012 06:30:33 +0000

Hi, all. I am new in this field and is facing some problems with the protein expression in PIchia pastoris system. I already cloned my gene into pPICZB for intracellular expression of the protein. Integration of the gene was confirmed by PCR and sequencing. Protein expression was carried out as suggested by the kit manual and protein expression was analyzed by SDS-PAGE and western blot. In SDS-PAGE, I saw protein overexpression in one band of more than 50 kDa (my protein is ~54 kDa) which correspond just correctly to my target protein. However, western blot with his tag antibody showed signal more than one band, the highest reactivity is with a band of more than 60 kDa. Apart from that, I tried to purify the protein and SDS-PAGE of the purified proteins showed single protein band of ~70 kDa. I'm quite worry whether is the protein is really my target protein. Could someone advice me? Thanks.

Problems with detergents and Ni-NTA

Tue, 31 Jan 2012 07:36:56 +0000

Hi all,

I am working on a GPCR which is expressed as an insoluble (not inclusion body) precipitate by cell-free protein synthesis.
My current work flow involves expression, resolubilisation, functional assays and/or purification. To date I have had no issues with resolubilisation (I get complete resolubilisation in <10m minutes at 30 degrees C with shaking).
I reolubilise into the detergent 14:0 lyso PG (LMPG) at 21mM (1%) link below. CMC is quite low, around 0.05mM
Unfortunately upon application to Ni-NTA I loose the majority (>90%) in the flow through. This is regardless of the pH (7.5 or 8) and so far regardless of the dilution of the detergent to 0.1mM by increasing the volume of the sample (eg: starting volume of 1mL made up to 200mL)

Ni-NTA binding buffer is 20mM NaPi pH7.5 or 8, 150mM NaCl, 5mM imidazole plus protein/detergent

What I do recover is a very small fraction and is unfortunately of no use to me at such small quantities.
Could someone please help me with any options that avoids complete denaturation.

http://avantilipids.com/index.php?option=com_content&view=article&id=731&Itemid=189&catnumber=858120

Additional info: I wish to bind to Ni to perform detergent exchange with DDM

Cheers,

PaddyS

Missing expression of proteins of interest in Pichia pastoris

Mon, 30 Jan 2012 19:46:54 +0000

Hi all

Hope that some of you might be able to help me out - I am seriously frustrated!
I am working on expression of two different plant lipid transport proteins (LTPs) in Pichia pastoris. They have a size just below 10 kDa. I have cloned the sequences into the pPICz(alpha) A vector from invitrogen (secreted expression) and confirmed the success of this step with sequencing of the constructs. Then I have transformed the linearized vectors into P. pastoris X33, and confirmed that the resulting transformants posses resistance to Zeocin. Furthermore, I have used different PCR screenings to confirm the correct integration of the constructs into the pichia genome.
So far so good.
Then I have chosen one transformant for each construct which has shown positive PCRs. I have performed expressions according to the Invitrogen protocol: First I grew up the cells in BMGY media, then shifted to BMMY media with induction with methanol every 24 hr and 250 rpm. I have tried growing them for 96 hours and 72 hours, and I have tried varying the temperature (23-29 degrees). The cultures seem to be growing just fine at all these conditions, and I have previously expressed (bigger) proteins in Pichia, using the same condition and system, without problems…
Furthermore, I have no antibody available to do a Western blot.

So now for the current problems:
1) I seem to have a very low general amount of protein, both in the background culture and the transformants. In lyophilized supernatant from background and transformant cultures I only have approximately 0.2% total protein present in the powder. Furthermore, I almost can’t see anything on SDS PAGEs, even when using silver stain. Bands are only visible at the top of the SDS PAGE, which then gets more and more unclear, and ends up in a smear.
2) I cannot confirm the presence of the proteins of interest on an SDS PAGE –possibly because they simply are not there. I have been using 10-15% glycine SDS PAGEs. I am soon to try a tricine SDS PAGE, but I believe I should be able to see something on the glycine SDS PAGE?!
3) IF the proteins are not expressed – how can this be? Is it possible that expression just does not occur, even though everything seems to be correct? Any suggestions what to do now?

I am in a bit of a pickle… I have a very limited time schedule to get success with this expression; else I have to move on to another project. Therefore I hope that someone can give me some good advises or hints 

Thanks in advance!
- Katrine

TEV purification unspecific band appearing

Wed, 25 Jan 2012 17:21:12 +0000

Hey guys ,
i'm writing because every time i'm doing His-TEV purification, when I check the induction a non specific band comes up in the coomassie gel.
His-TEv is 27kDa, this band is around 40-42 kDa.
I would exclude it's a dimer. Do you think it could be a glycosylated form?

Thanks for your help

No protein expression after galactose induction

Wed, 25 Jan 2012 09:13:02 +0000

Hi all,

I am having problems with getting expression of my genes which were cloned under the GAL1 promoter of pYES2/CT plasmid from invitrogen.

I had first inoculate the transformed yeast colonies in YNB -URA medium with 2% raffinose overnight. Then after around 12-16 hours, I transferred the cell pellets to the induction medium which is YNB -URA with 1% raffinose and 2% galactose. My cells were growing normally with OD600 peaked at a reading of around 13. I had collected samples from the induction medium from 19 hours to 71 hours after induction. Samples were taken every 12 hours after the 19th hour of induction. I tried to run western blot and even RT-PCR but all tests gave me negative results. My genes were successfully transformed inside the yeasts as confirmed by colony PCR. Thus, something must be wrong from the induction steps onwards.

I read it from somewhere that galactose induction occurs as early as 2 hours after induction. Is it true?
If true, then is it possible that by the 19th hour, all the galactose were used up and thus I am getting negative results?
Hope someone will be kind enough to offer some advices on this. I had be trying at this for nearly 2 months now. Any help will be greatly appreciated. Thank you in advance.

Cheers,
KY

Purification of Sequence-Specific DNA-Binding Proteins by Affinity Chromatograph

Tue, 17 Jan 2012 22:32:20 +0000

I want to make an oligonucleotide for Purification of Sequence-Specific DNA-Binding Proteins by Affinity
Chromatography. I just want to know is there any tips and tricks for DESIGNING the oligonucleotide like designing Primers? for example
how long should it be?
How many times the Sequence-Specific DNA should be repeated?
How long the flanking DNA should be ? and its content? CG rich or AT rich?
Do Nucleases interfere and digest my immobilized oligo-nucleotides?
Which way is better for coupling the oligo-nucleotides to resin and which resin is better? Cyanogen Bromide is better or binding of biotin to streptavidin?

how to choose the best Sequence-Specific DNA and which websites can help me?

I would be appreciate...

Protein extraction from mouse feces

Fri, 13 Jan 2012 09:31:55 +0000

Hi,
I would like to extract protein from mouse feces. Could you give me a protocol for that? And did you know the amount of protein can I obtein from this extraction?

Thank's in advance

DoD

Proteominer on VERY Low Protein Levels

Wed, 11 Jan 2012 15:52:56 +0000

Hi all,

I use Proteominer Enrichment kits on serum samples all the time and recently I've started working on vitreous fluid. Unfortunately, both the sample volume and concentration of the fluids are low so I can't process them using the ordinary kits.

I would, however, like to try and use a small amount of beads, relative to the sample concentration and volume, from the column to process the samples. Initially I plan on optimising this for 2 samples. The total concentration of one is 2.23mg and I have 250uL. The other sample has about 0.4mg and I only have 120uL of this.

I understand that this is difficult task but it would be great if I could at least try this and see the results. Does anyone have any tips or ideas?

Thanks.

Bradford method

Wed, 11 Jan 2012 11:32:41 +0000

Im sonfused about :

Which step that significantly reduces the yield of your product protein?
What are the important factors in performing Bradford assay correctly?
Is BSA good to use for my standard curve ?

Thanks for ur help !

protein extraction from pirmary keratinocytes

Tue, 03 Jan 2012 11:24:11 +0000

Hi!
I've been trying to isolate proteins from human primary keratinocytes using RIPA buffer, but every time i get very low concentrations (ranging from 0,8 ug/ul to 1,5 ug/ul). I do it by trypsinising the cells, harvesting them to an eppendorf tube, centrifuging and suspending the pellet in RIPA.

I have never encountered this problem before with other cell lines.

I've modified the the protocol I had been using (changed the centrifugation speed from 1.200 rpm to 2.000 rpm, use different eppendorf tubes - the result improved a bit, but is still insufficient). The low concentration results in huge solution volume I need to apply on electrophoresis gel and I think that it causes problems with protein detection (with HRP-conjugated antibody).

Do you think that this low concentration may be really the cause of detection problems (uneven, dot-shaped or beads-shaped bands)? I use high antibody concentrations and long exposures to detect my protein of interest, and when it shows, the blot looks quite awful and is not suitable for any interpretation.

Do you have any ideas how I can improve the protein concentration?

How to purify unstable protein?

Fri, 30 Dec 2011 10:17:02 +0000

I am facing problem with purification of a mutant protein by E. coli expression system. Expression of that soluble protein is good and like as wild type. But after purification of that protein by Ni-NTA column, when I tried by gelfiltration it was denatured. How can I solved this problem? I have purified wild type with same procedure without any problem. Please suggest me. Thanks in advance for your suggestions.

unstable flag tagged protein

Thu, 22 Dec 2011 10:54:58 +0000

Hi
I used the expression vector pCDNA3.1/flag to subcloning the C-terminal region of BRCA2 and transfect it into BRCA2 deficient cells. After Immunoprecipitation and Western blotting analysis with a flag antibodies, I didn't see the expected band of approximately 80 KD but two bands of 50 and 30 KD.
Any suggestions?
Thanks in advance
Laura

Hisx18 purification

Wed, 21 Dec 2011 23:36:17 +0000

Hi Folks,
I am trying to purify a protein with 3 Hisx6 tags. Any ideas on how high I can go with imidazole in the washes without losing my protein? I am getting loads of background even under stringent conditions (6M Guanidine HCl or 8M Urea). I think the trouble is that I am purifying from yeast and yeast has a lot of histidine rich proteins. Any ideas or suggestions?
Thanks.

Streptactin sepharose purification- binding problem

Fri, 16 Dec 2011 12:27:12 +0000

Hi,
I am trying to purify an archaeal protein expressed in E. Coli so I am using high salt buffers:
Lysis & wash buffer 2M KCl, 100mM Tris
Elution bufffer 2M KCl, 100mM Tris,destiobiothin 2.5mM
All buffers at pH 8.
I am using IBA Streptactin Sepharose and gravity flow colums.
I did one purification as described in the IBA manual and washed column with 6x1ml of wash buffer.
However, I got my protein and other that bind not specifically so I decided to do a 2nd purification and wash the column 2x10 ml of wash buffer.
In both cases I eluted with 6x 0.5ml.
Before the second purification I incubated the sepharose resin with lysate for one hour with rotation.
While in the manual they say not to do it I think in this case this could not have caused problems since the high salt would already inactivate proteases.
The problem is that when I washed the column more I did not recover anything. (no bands on SDS-PAGE)

I can't think what might be the cause of this.

Thanks for any ideas

remove antibody from eluate in co-immunoprecipitation

Wed, 14 Dec 2011 22:51:39 +0000

Hi!
I'm running a co-immunoprecipitation experiment in which I used quite a lot of protein extract and Flag-agarose beads. I eluted with the flag peptide but even with this I know I have quite a lot of antibody eluted and present in my sample. Before loading the SDS-PAGE for cutting bands for MS analysis I would like to remove the antibody. Anybody know a good way to do this? I'm affraid the IgG will be very intense in my gel after staining and will hide other proteins. However, I don't want to manipulate my sample a lot to avoid protein losses. Any piece of advice is welcome!!

Thanks!

How to obtain high-level production of low molecular weight recombinant proteins

Wed, 14 Dec 2011 08:39:40 +0000

I'm going to cloning and overexpress a 7.5 kDa protein in E. coli base on pET vector system. But the problem is the low molecular weight proteins rapidly degraded by E. coli proteases. What should I do?

Sonication - to completely lyse cells

Fri, 09 Dec 2011 07:32:47 +0000

Hello there,

I am using this sonicator for first time - It has setting for pulse time & amplitude %.
I have expressed protein using BL21DE3 cells - I want to lyse the cells completely to seperate the soluble and pellet fraction - please suggest the amplitude and time for pulses
previously i used 50% and 5 pulses of 10 sec but the cells did not lyse completely - almost similar protein pattern was visible in both soluble and pellet fraction.

Thank you very much for the help

Using the recombinant/fusion protein for polyclonal antibody development

Thu, 08 Dec 2011 17:43:46 +0000

Hi all,

I know this is a dumb idea, but I have to ask ....
I have three genes cloned into pET32a+ vector and the protein ( lets say fusion protein-Trx gene +gene of interest) expression in Bl21Plys is good (the protein i get in native conditions and not in inclusion bodies at all) . Purified using His tag columns, that too went fine...now comes the real mess I tried to remove the tag that is the Trx tag using enterokinase ,the protein gets digested with the fusion protein and protein of interest almost running together in PAGE, but not purified using the his tag columns.....
I am running short of time for my thesis work , the real question is has anybody used the trx tagged fusion protein for development of polyclonals in rabbit????
I know its not the real sense to use the fusion protein for polyclonal development, but has anybody tried to do so??

Thank you all.....

Insoluble Protein

Thu, 08 Dec 2011 12:23:07 +0000

I got this protein and I really wanna produce a reasonable quantity for protein crystallization.

I grow the cells 37C then induce it with 400uM IPTG. Harvest, lyse and run a gel but I don't see any band at all in the lysate. Band is clearly on the pellet.

I checked the sequence. The GRAVY index is 0.14, which I don't think is really terrible, since I got some constructs which have index higher than that (0.38) and produce very well as soluble protein.

Any suggestions? I don't really wanna go to the route of mutagenesis as much as possible.

Would lowering down growth temperature and lower IPTG do the trick?

Thanks!