Protein Purification - Science method

I have a plant gene which is cloned in pGEX4-T-2 vector.

I facing problem during protein purification. the proteins was getting out in flow throw and wash.

Lysis buffer contain 1XPBS + lysozyme + PMSF + DTT 1mM

Elution Buffer contain- 50mM Tris HCL + Glutathione reduced

Hello.

I am working on heterologoulsy expressing and purifying a protein using the Bac-to-Bac method. The protein expresses as verified by western blotting. I typically do a 4.2 L expression using P2 baculovirus. I lyse the cells via sonication in the following buffer (5 mL per gram of wet cell mass): 20 mM Tris pH=8, 500 mM NaCl, 10 mM imidazole, 5% glycerol, DNAseI, and protease/phosphatase inhibitor tablets. After sonication, I centrifuge the lysed cells for 1 hour at ~54,000 x g. After centrifugation the supernatant is not viscous and has a clear yellow tint. This clarified supernatant will clog the IMAC column (I have tried both HisTrap and self poured columns). Only when I filter all of the clarified supernatant using a 0.22 um syringe filter can I load without clogging. Does anyone know a better way to solve this issue of column clogging?

Hi All,

I wanted to purchase Rosetta™ 2(DE3)pLysS Cells (Product No# 71403-M )from Merck but I was told that this product has been discontinued. In the circumstances, could you kindly suggest/recommend me an alternative strain to Rosetta™ 2(DE3)pLysS Cells?

This strain is intended to be expressing the LbuCas13a protein (at 16 degrees Celsius) and the capsid protein of tobacco mosaic virus (TMV).

Thank you for your time and consideration.

I look forward to hearing from you.

Subha

Good day everyone,

Trying to perform protein purification for the first time, gotta buy the whole equipment/reagents.

Protein of interest 48 kDa (Antithrombin), expressed from his-tagged plasmid in culture cells. Unfortunately it's secreted in low amounts from the HEK cells, thus going to extract it from approx. 15 ml medium.

Ni or Iron his-tagged beads? Alternatively, resins? Any specific brand/model?

Will need also a magnetic rack for falcon tubes

I have zero idea while so many options on the market, too much confusion.

Any recommendation highly appreciated!

Hello everyone. Long story short, I am struggling to purify a soluble protein which has a 6X His tag. I ruled out the issues with the expression vector, as well as faulty induction (i.e small scale expression went fine and showed up on the SDS PAGE).

I elute the protein with Imidazole 250mM using 3 buffers with varying pH and the gel shows that it gets stuck on the Ni resin with no protein at all (not even faint bands) in the elution fractions. The protein is not too stable so I don’t want to experiment with pH a lot. Should I increase the concentration of imidazole? What is the reasonable concentration of it for elution which won’t complicate the further purification and quantification (BCA assay will be used).

Thank you very much!

I am purifying RNAse E in E. coli (118 kDa). I cloned the rne coding sequence on pET28a with a His tag at the N-terminus. The protein overexpressed fine (see attached figures). The binding buffer contains 20 mM Tris-HCl (pH 7.5), 300 mM NaCl, 1 mM DTT, and a proteinase inhibitor. The elution buffer contains 20 mM Tris-HCl (pH 7.5), 300 mM NaCl, and 250 mM Imidazole. I tried binding at both room temperature and 4 degrees Celsius for 1 hour to overnight, using low and high salt concentrations in the buffer. However, the amount of protein that bound to the beads was very low. I have performed several His-tagged protein purifications before, but I have never experienced this issue.

Hi, may I know

Thank you for any assistance!

I am trying to recharge the Ni-NTA column for his-tagged protein purification but my NiSO4(H2O)6 is not dissolving in 1x PBS and I have tried it from a different reagent bottle also, please help!

pI of my protein is 4.1. I am using Ni coumn to purify it . Which binding buffers will be good?

Even the pI is low can it still bind to nucleic acid? How do I know?

Hello esteemed researcher, May I direct your attention to the image? I am curious to understand why there is only a minimal drop in pressure once the elution begins.

thank you.

I am trying to express a FAD containing enzyme of mycobacteria in E.coli. I am able to purify the protein which is slightly yellow in color but it seems that my FAD is all unbound to the protein. How can I express the protein which has tightly bound FAD?

Hi,

I saw a picture on a web (https://www.chegg.com/homework-help/questions-and-answers/9-calculated-molecular-weight-native-gfp-denatured-gfp-native-denatured-proteins-differ-mo-q53517323). It show two computational formula for native and denatured GFP protein respectively. But I can not understand the behind mechanism... Is it a trusty information? Or dose anyone know about this and provided some help?

The SnapGene show that EGFP with 239 amino acids and is 26.9 kDa. But the picture showed 28.183 and 31.622 kDa respectively. That's strange....

Thanks,

Best

I am currently trying to purify proteins from different insect cell lines and baculoviruses. One of the criteria I want to check is the amount of host cell protein (HCP) in the purified sample. However, while there are ELISA kits available for Sf9 & Sf21 cells there is no such kit for HighFive cells.

Does anyone know of a kit, or an alternative method?

We are interested in buying a new protein purification system for the lab. On the side of Akta and BioRad, we also found the Autopure system from Inscinstech. Did someone already try this protein purification system? Do you have advice/recommendations regarding the AutoPure equipment or Inscinstech company?

Hi everyone,

I have been using MagneHis™ Protein Purification System (V8500) for many years to purify proteins from insect cell culture, Baculovirus expression system. Currently, I have difficulty in purification using this system. I can detect my target protein in the cellular extract by western blot analysis using anti-his antibody, however, I can not detect it or is seen very sligthly, after purification, figure is below. I would be very happy if could suggest me an alternative strategy for protein purification. I would appreciate it very much for your kind help.

I use the following method to wash my inclusion bodies: 8 steps.

1: Troton, EDTA, Tris and Fresh DTT (3 Times)

2: Doc, EDTA, Tris and Fresh DTT (3 Times)

3: PBS

4: Water

I use this method for 3 different recombinant proteins. In 2 of them, host proteins were removed up to 90% during washing. But in the third case, a very small amount of host protein is removed and I have a lot of impurities.

Can someone tell me that

Do I need to change the concentrations of the compounds in the washing buffers?

Do I need to choose another method?

Choosing an IB washing method depends on what factors?

Thanks a lot.

Hi there.

I have to use the pET28a-SUMO vector to get my target protein.

However, I can‘t separate it from SUMO tag.

My protein was about 15 Kda big and has a about 5 value of pI.

After tag on/off-columns cleavage, my protein will stick on the Ni column if I want to remove the tag only if I introduce buffer with imidazole. But buffer with imidazole will bring the SUMO tag down even you use 10mM imidazole.

How can i get my protein without SUMO tag ??????

I have tried ion exchange chromatography and It was not work.

6His tag only? Inclusion body.

MBP tag?TEV is trash enzyme and same problem when i remove the tag.

GST tag?A little of protein is soluble.

Currently I am working on beta-lactamases. I am using pET-28a vector(present kanamycin resistance gene) for cloning purpose, and transformed in E.coli BL21(DE3) bacterial cells. During purification, primary culture looks fine and there is uniform bacterial growth but in secondary culture(both times use kanamycin at concentration 50ug/ml according to CLSI guideline) filamentous type of growth is coming, surprisingly OD increases very slowly that time. I have rechecked the autoclave procedure, and freshly made kanamycin. Even after that same circumstance.

What could be the possible reasons?

Could you kindly provide insights into suitable techniques or protocols for achieving effective protein fraction drying following acetone precipitation, considering the limitations associated with freeze drying? Your expertise and recommendations in this matter would be immensely valuable to the progress of my research.

Suppose I want to prepare 0.1M sodium citrate buffer of pH 3.0, I observed that most people use citric acid monohydrate (as weak acid) and sodium citrate dihydrate (as sodium salt of weak acid) why?

Citric acid has three pKa’s:

pKa1 = 3.13

pKa2 = 4.72

pKa3 = 6.40

Our desired buffer pH (0.1M sodium citrate buffer) is 3.0 which is close to pKa1 = 3.13, So we have to use citric acid monohydrate (as weak acid) and sodium citrate monobasic ( as sodium salt of weak acid) instead people using sodium citrate dihydrate (as sodium salt of weak acid) why?

Is there any specific reason for using sodium citrate dihydrate rather than sodium citrate monobasic (as sodium salt of weak acid)? If so then among three pKa’s which pKa we have to use in Haselblach-Henderson equation to calculate weight’s of each component to be added?

Hi All,

We routinely use HEK293 cells to produce recombinant proteins and antibodies in our facility and we observed that for a small portion of proteins/antibodies, there appear to be a non-protein type of aggregates that co-elute with the target protein/antibody during purification. Such aggregates can be visualized as precipitates that have a string-like appearance and can be removed by centrifugation. The removal of these aggregates doesn't alter the protein/antibody concentration and they will re-appear after 4C storage. I was wondering if anyone else might have faced a similar problem and I would appreciate any suggestions on how to remove or prevent such aggregates from forming.

P.S. We use a culture system similar to that of EXPi293.

Being a biotechnologist, I am always curious to know the real example where a stakeholder shows enthusiasm and implements an algal consortium or axenic culture to treat raw dairy wastewater (RDWW). If you are planning to implement this system, then you may face many barriers, like:

1. temporal change in RDWW

2. unsterile RDWW has its microbiota; maybe it competes with the algal consortium

3. Which cultivation system does it adopt: an open raceway pond or a closed system?

4. the flow rate of incoming RDWW.

6. Which mode of cultivation is more suitable for real-time RDWW treatment (batch or semi-continuous)?

7. After or before, which kind of operation enhances the remediation capability and is ready to use or discharge in natural water bodies?

8. How do you maintain the C/N/P ratio?

9. What about the smell of RDWW due to protein purification?

Hello,

We are trying to purify proteins using a secretion system but do not have TFF cartridges for our device. Does anyone know where we can purchase these? Or is there a better replacement? We want to downscale the volume from 3 L to 100-200 mL.

I've attached a picture for reference.

Thanks,

Thomas Newton

Hi all,

I am a freshman in protein purification. I found an interesting fact!!!

After elution by buffer containing peptides which can competitively bind resin for protein, target FLAG-protein can be gathered. So I think peptides should be captured by anti-FLAG resin. However, I found that when I continued another batch (same protein) of purification, the protein still could be enriched on this anti-FLAG resin with a similar binding efficiency. Actually I didn't wash with an acid solution or do resin regeneration before the second time I used it.

That's confused me a lot. Why resin still can be used without resin regeneration but without losing its ability to bind to the protein? How do peptides disappear on anti-FLAG resin?

Similar to Ni resin, we found resin can be used several times without EDTA regeneration treatment. How does imidazole disappear on Ni resin?

I really appreciate your answers or suggestions~~~

I am trying to purify a N-terminal his tagged secreted protein. The protein of interest is a self assembled nanocage and is engineered to express his-tag on its surface which enables downstream purification. It is essential that the his tag be appended on the N-terminal for surface expression. I am unable to pickup any signal with western blot analysis using anti-his antibody (western control works). I read somewhere that hydrophilic tag (his) can affect cleavage of hydrophobic signal peptide (CD5 leader sequence). Could this be the reason? The recombinant protein was cloned in the following sequence; CD5 signal peptide-linker-6xHis tag-linker-protein of interest. The linker used was Ser Gly Gly. Any tips will be greatly appreciated!

Good afternoon,

I try to extract cell wall proteins from green macroalgae, I have tried different protocols and one of them included step of EGTA extraction, however this fraction is jelly like, so I expect polysaccharides from cell wall to be present. When loaded to SDS-PAGE, it was obvious that the gel pores were blocked and no bends were obtained, just light blue streak. According to Bradford, in this fraction I have quite a lot of proteins with which I would like to do Western Blot... I have already tried aceton precipitation, but still a lot of polysaccharides remained....So please do you have any recommendations how to get rid of these polysaccharides?

Thank you very much,

Tereza

Hello.

What is the correlation between protein denaturation and its hydrophobicity?

What is the effect of changing the hydrophobicity on retention time (reverse phase chromatography)?

in my protein, the retention time has decreased by one minute, is this amount normal?

Thanks

I expressed the protein(46kDa, PI=9.04) with His tag in E.coli and purified it with cobalt beads. However, when I was using Amicon Ultra 15 mL Centrifugal Filters to concentrate elution flowed through the cobalt column, protein aggregated. The whole process is on the context of 4 degrees celcius and buffer is 20 mM Tris, 150 NaCl, pH7.5. Could someone give me some suggestions to solve protein aggregation?

Why does a solution of 50mM Tris and 20mM CaCl2 sometimes precipitate?

This buffer is used for protein purification.

Hello Everyone,

I’m exploring the feasibility of a mobile application that assists in identifying contamination in microbial cell cultures. The concept involves the following steps:

The application, using deep learning algorithms, would analyze the shape and color of cells to detect patterns indicating contamination. Users would need to provide specific details such as:

I would appreciate your insights on the following aspects:

Thank you very much for your valuable feedback and time!

Hello, I've been doing protein purification more in the lab and I want a better understanding of the technique. I've been looking into it on my own but was wondering if anyone knew of a good resource maybe I haven't found yet. Thank you!

Hello,

I am trying to express the VHH that is discovered in our lab. The isoelectric point of the VHH is 7.75 and I tried using pH of 8.00 and 6.8. The gene of interest with His tag is inserted in the pET22b(+) vector and is transformed into BL21(DE3). I use IPTG to induce the protein. The gel ran for pre and post-induction samples shows no difference between pre and post-induction bands. Assuming the VHH expression is very low, I purified the protein with IMAC and SEC. The bands below in the image are the bands from concentrated protein after SEC purification. I also tried protein purification with ammonium sulfate precipitation. It did not help that much and the protein expression is significantly low. My question is why do I see multiple bands with high molecular weight even after SEC purification? And also, why the protein expression is very low? I appreciate all the answers and feedback.

Thank you.

Sashi

The purified protein is exhibiting a trimeric state based on my NATIVE PAGE gel and SDS PAGE shows monomeric configuration. Can you recommend detergents to destabilize the H-bonds mediated by sulfate ions (based on literature)? I have already tried BME and DTT and they don't work.

I have been purifying proteins which have an N-terminal His-tag using Ni-NTA affinity chromatography. I carried out size exclusion chromatography using Superdex HiLoad, Superdex, and Superose columns. I also carried out dialysis of the Ni-NTA purified proteins.

Both the above experiments were performed for buffer exchange and to remove non-specific proteins.

When I ran SDS-PAGE gels for the Ni-NTA purified and dialysed proteins, I could see a single band corresponding to the protein of my interest. Multiple bands beneath the protein of interest could be seen on SDS-PAGE gel for the SEC fractions.

I used appropriate controls to rule out the possibility of degradation due to prolonged exposure at room temperature and the effect of varying salt concentrations.

Please help... to all the protein purification experts out there!!

Hi

I'm purifying some mutants of the protein I study. The wild type protein exists as a monomer and is 28kDa.

I have two mutants (same protein, same number of amino acids but with 8 amino acid substitutions at defined positions), one of the mutants (mutant 1) analysed using size exclusion chromatography with multi-angle static light scattering (SEC-MALS) and its MW was shown to be 33kDa and has an oligomerization state of 1.2. The other mutant, mutant 2, also measured by SEC-MALS was 59kDa with an oligomerization state of 2.2.

For the wild type to measure the concentration I've just been using the MW (28kDa) and extinction coefficient (calculated by entering the sequence into online software ProtParam) and using a NanoDrop measuring absorbance at 280. This gives the concentration in mg/ml which I then convert to molar concentration.

For the mutants I want to measure their concentration the same way - measuring A280 on the NanoDrop using the mutants MW and extinction coefficient and calculating molarity from mg/ml. I'm not sure if this is an obvious/stupid question but what MW weight and extinction coefficient would you use for the mutants on the NanoDrop? E.g. For example mutant 2 molecular weight (MW) of the protein based on its amino acids (AA) composition is predicted to be 28kDa, but SEC-MALS shows it is 59kDa as the protein forms an oligomer.

My instant is to use 59kDa and the computed extinction coefficient predicted from the AA composition - is this correct?

Thanks in advance!

I use the refolding buffer with the following specifications for the refolding of brolucizumab protein.

l-Argenine, Sorbitol, EDTA, Tris, and fresh Cystein and Cistine.

I have already used these compounds to refold this protein and I was getting a proper protein refold. Currently, although I use the same compounds and with the same concentrations, the answer I get at the end is not the same. In fact, the protein disappears after being solubilization and added to the refolding buffer.

The issue really confuses me and I don't have an answer for it. Can anyone have an answer for me?

Earlier I had used Serva IPG Strips in which it was easy to decipher the gel side of the strips but recently we shifted to Bio Rad and here we are encountering this problem. Your valuable suggestions are required.

Dear All,

I am trying to purify a protein secreted by HEK293 cells. My target protein fails to specifically bind on a nickel/cobalt column probably because of the presence of BSA coming from the serum. The protein is eluted at 50mM imidazole along with a huge amount of BSA.

Moreover, my target protein has very similar isoelectric point and MW than BSA so it is getting very difficult to separate them using classical methods.

Any advice ? Is there a specific resin that is binding to BSA ? Or something that can be added to reduce the non-speficic binding of BSA ?

With many thanks,

Gianluca.

I do an 8L induction at 18C for 16-20 hours in TB media. I routinely harvest ~50-80g of bacteria. I freeze my pellets at -20 for > 3 hours after harvesting, and have been using a 1kW blender to resuspend and homogenize them in my lysis buffer. However, I have been noticing that the blender causes a lot of air to be entrained in my lysate, which seems to be affecting my protein yield. Is there a better method to resuspend and homogenize my quantity of bacteria?

I can provide more information upon request.

Ammonium sulfate (40%) was used to precipitate proteins from calf thymus extraction. After centrifugation, the pellet was dissolved in PBS. The pellet can’t be dissolved completely (still had lots of pellet after centrifuging at 12000g for 10 min).The supernatant was cloudy and couldn’t be filtered through 0.22 um NC membrane. We further centrifuged the supernatant at 12000g for 1 h, but the supernatant was still cloudy and could not get through 0.22 um membrane. To get clear sample through 0.22 um NC membrane is the first step to further purify the proteins. Now it’s totally stucked here. Is there any recommendations? THX.

What are the steps in the regeneration of 'fresh' DEAE Sephacel, which comes in the swollen form, in 20% ethanol. And what is the significants of each chemicals in these steps, can anybody explain?

I have a trouble with my dialysis bag

Once protein has been extracted and quantified, and before protein digestion, I even concentrations to 1 mg/ml using U/T buffer (7M urea, 2m thiourea, 30mMTris). Then I re-quantifiy protein to make sure that protein concentration is 1mg/ml. For some reason that I do not know, buffer and protein do not mix homogeneously; the more concentrated the original aliquot is and the more buffer is added,  the less concentrated the final aliquot is. I did repeat the protocol 3 times with fresh buffer and I always get the same. However, I did not have this problem when sample was diluted in water. Any idea or suggestion? Thanks!

I'd like to perform a SEC step prior to on-column refolding of a protein I am expressing/purifying but am worried about the extended time the protein will be in the presence of urea (and subsequent protein carbamylation) in the current refolding workflow I have.

Is there any concern of protein modification with a 4-6 hour denatured protein purification workflow using 6M GndHCl?

Hi everyone

I have a problem with Histag protein purification. I have this construct with his tag on N-terminal. I went for small-scale expression and I had some good bands. However, on a large scale, the protein didn't bind with the HiTrap TALON cobalt. My protein contains an Mg2+ ion coordinated by the side chains of Asp, His. How to overcome this metal ion issue, or Should I change the column to a Ni-Trap?

I appreciate your help

I am purifying a protein by denaturation method(8M Urea). After solubilizing and running a His TRAP with 1M NaCl Wash, and elution. I refold by dialyzing to 2M urea and then desalting the column to remove Urea and refold. At lower concentration till 1 - 1.5 mg/ml, I have a normal 260/280 of around 0.63 but when I concentrate further the protein starts showing weirdly higher 260/280 (like <1 ). I see no precipitation also( 350 nm seems fine and visually also protein is not cloudy). I am quite confused with this trend. Does anyone have any similar experience?

How can I overcome this problem?

When measuring protein concentration with a spectrophotometer, is the use of Coomassie Brilliant Blue necessary? Protein (BSA) has a peak in UV, so can I prepare different concentrations of it and determine the unknown concentration?

Currently I am doing protein purification from my transformed E. coli by using Histrap FF column. The protocol says, to prepare the sample I can dilute it with binding buffer but not using strong bases/acids due to precipitations risk. In my lab we only use NaOH and HCl for pH adjustment. What weak bases/acids usually used for pH adjustment?

I am trying to express the soluble fraction Membrane Protein SARS Cov2 in E. Coli. I am getting only monomers while it's the dimer form that is active. Literature that I have come across have used yeast and mammalian expression systems only. I want to know if it can be expressed in E. Coli. If not, why?

Why does the initial protein elution (elution No 2)(Each elution volume 500microL) in maltose binding protein purification exhibit high concentrations of the target protein on SDS-PAGE, but fail to demonstrate high activity compared to the lateral elution fractions with lower target protein concentrations? Can the activity of the target protein be disrupted by the expression of other proteins related to the vector (PMALC2X)?

I am currently trying to express Turritopsis dhornii TET enzyme with a 6x HIS tag in a BL21 E.coli host. Every time I purify my protein it shows double bands on SDS-PAGE, when testing its activity on ELISA there seems to be no activity. My lysis buffer contains 50mM HEPES ph 7.5, 30 mM imidazole, 500mM NaCl, and 1mM DTT. I purify with 800uL of Nickel Sepharose beads. Are there any adjustments I could make to stop this double band from showing?

By keeping rest of the protocol same does centrifugation speed will have any effect on protein extraction from XL1-blue colonies

What is the Maldi-tof technique?

Is it necessary to perform Maldi-tof technique after protein purification?

My N -terminal GST tagged protein fail to purify using this condition: 30ml sample (3mg of crude protein) injected at flowrate (0.5ml/min), 40ml wash (PBS pH 7.3) and 5ml elution (50mM Tris HCl + 10mM reduced glutathione, pH 8). For washing and elution, the flowrate was 1ml/min. Buffers were prepared all according to the manual. For your information, based on SDS -PAGE analysis, the crude has decent amount of GST tagged protein. Therefore, I suspected that the GST tag was hidden in the conformation. The protein structure was not discovered yet so I can only hypothesized. Based on previous structural analysis, the N -terminal was predicted to be at the cytoplasmic region. Does this means, whatever tag that I put in the N -terminal will be forever hidden in the structure? Should I cleave the tag and purify the protein using other means (IEX/ SEC/ HIC)?

suppose: we have a purified protein with us; after this step what are the methods and procedures that can we use to see the protein is intact- KINDLY HELP

The purpose is cell based functional assay for the recombinant protein not protein purification.

I have laryngeal cancer samples from which I am to extract DNA/RNA and Protein. However, the kit I have only extracts DNA and RNA (Qiagen AllPrep DNA/RNA Kit), and does not extract protein. How do I go about protein purification without having to order another kit, keeping in mind that the samples are quite limited in size. Thanks.

Theoretically, if the value of A260/A280 goes high it shows DNA contamination in protein sample, and if goes down (0.6-0.8) it shows good purity of protein. In this way, a value less than 0.6 should show a more pure protein. but lower value of A260/A280 of protein <0.5 is considered not good for protein purity. Why

I am expressing a human RNA binding protein in E. coli and purifying it using ammonium sulfate precipitation followed by heparin, butyl, and size exclusion chromatography. While the first batch of protein purification did not show any RNA contamination, subsequent batches consistently exhibited RNA contamination. I have tried to maintain the same purification protocol but cannot seem to eliminate RNA contamination.

Have other researchers experienced this issue while purifying RNA-binding proteins, and if so, do you have any suggestions for troubleshooting or improving protein purity? I would appreciate any insights or recommendations to help me achieve a pure sample of my protein of interest.

Dear researchers, I am now trying to express several proteins from bacteria（Agrobacterium Tumefaceins) in one of its biosynthetic gene clusters. However, some of the proteins can be easily expressed and purified but many of them can not even be expressed. I also checked the pellet by SDS-PAGE and found that there were no over-expression bands in the pellet.

I want to ask how can I possibly get these proteins? Here are my protein expression conditions:

vector： pET28a

host：E.coli BL21

Tag：6xHistag

Culture until OD value reaches 0.6 and 0.5 mM IPTG was added(working concentration) to induce over expression.

Some of the proteins in the cluster can be expressed very well using this common protocol, yet others remain even unexpressed. I do not know what had happened, since there were no over expression bands in the pellet, suggesting over expression did not even take place.

I tried other recombinant tags like SUMO, MBP and GST, but none of them helped.

I am really stuck in this situation now. :(

Hello

I'm sure this question has been asked a lot but the protein I am purifying is not as clean as I would like and all the potential solutions I have read about have not worked for me.

I am purifying a protein that forms inclusion bodies. The construct is 14x His tag - Tev cleavage - protein of interest. I use BL21 Star (DE3) cells and TB media for growing the bacteria and induce at OD 0.6 for 4 hours at 30 degrees.The purification protocol is based on a previously published paper. Following sonication, lysis buffer and washing the pellet the protein is extracted from the inclusion bodies using 6M Guanidine Hydrochloride, applied o/n at room temperature to a Ni NTA agarose column. It is then eluted in 4M Guanidine. I then pass it through a RP-HPLC. As you can see by the attached image although I am getting a high amount of protein it has a large smear which I am not sure if it is contaminants or degredation. I overloading with sample when trying to judge purity but I think it's better to get an accurate representation of what’s going on rather than kid myself I am working with a pure sample.

The HPLC makes no difference so I think optimisation on the Ni-NTA purification is needed but nothing thus far has worked, I have tested different induction temperatures, leaving it on the Ni column at both 4 degrees and for a few hours (rather than O/N at RT like the protocol says), protease inhibitors and washes/step-wise elutions with different concentrations of imidazole (as in attached file) - yet none of this has worked. I've even thought about making the His tag smaller (14x seems quite large, but I cant see how this would impact purity other than needing more imidazole for elution).

If anyone has comments on the purity and has any tips that I have not tried it would be greatly appreciated!

Thanks!

Hey, I need help! I'm trying to purify two proteins (26 kDa and 96 kDa) using a Ni- NTA resin. Both enzymes have a 6- histidine tag (bioinformatic models shows that the tags are correctly exposed), and the expression occurs in E. coli (strain BL21). The problem I'm having is non- specific binding, the elution come out just as dirty as the original sample or the flow through.

I have tried using two different brands of resin (Quiagen and Cytiva), currently using the last one. I already tried using different buffers (20 mM Tris-HCl, 300 mM NaCl + 300 mM imidazole for elution, pH 8.4 or 20 mM sodium phosphate, 500 mM NaCl + 500 mM imidazole for elution, pH 7.4), just as indicated in the manuals.

I tried two methods of cell disruption (lysozyme + freeze and thawing cycles or sonication), thinking that maybe the lysozyme was interfering with the resin's maximum capacity. I've tried doing both -batch and column purification-, and got the same result in both cases. It puzzles me, because my mentor purifies many proteins from E. coli lysates using the same resin and conditions, and she gets good results. We don't know what's failing, I'm going crazy! Will it help adding a nonionic detergent to the elution buffer (e.g., 0.2% Tween- 20)?

These are today's results. I resuspended the bacterial pellet in 50 ml of binding buffer and sonicated it. I centrifuged and passed it through a 5 ml column, with a flow rate of 0.5 ml/min and usind an imidazole- gradient elution. (I already tried increasing the flow rate). I got a good peak elution, but when I load the collected fractions on a SDS- Page.. There are a lot of proteins, and they all elute together in one unique peak! (Sorry for the SDS- Page pic, I took it with my phone. Colected fractions are the last five ones).

Thank you in advance, Julieta.

I use 6x-his tag purification to purify my protein.After binding to the Ni-NTA beads, I use 6M urea washing buffer which contains 60mM imidazole to compete with the non-specific protein. However, my protein was washed out when I was washing the beads. Because of that, the elution quality of my protein are very poor. Does someone know the reason why?

I was looking for any recommendation for protein purification system for my research. We dont have much budget for akta and are looking for affordable alternative. We do have HPLC in the lab, can we attach column to the machine and purify our protein successfully?

Hi all,

I am a beginner in cryoEM field.

I know sometimes we must remove his, flag, strep, MBP, eGFP in X-ray crystallography study.

However, when I read papers, I found that these tags were not necessary to be removed in cryo-EM study. So I am wondering if we can keep all the tags for cryoEM study, especially the big tags such as MBP and eGFP tag, but will they affect the protein real conformation I mean in vivo conformation? I didn't remember who told me that MBP connects to proteins through a flexible linker, so I am not sure if we can see the big tag density in this case.

On the other hand, these big tags can help to increase the protein particle size, and may also help to improve the orientation preference in cryoEM study.

Are there any cases or publications that solved cryoEM structure containing big tags e.g. eGFP or MBP tag?

Any comments or suggestions are welcome, thanks!

I have solubilized my protein with 0.3% sarcosine and purified by using Ni-NTA,during purification most protein is going into flow through.

I have diluted my sonicated sample to 0.1% sarcosine but still I am unable to get binding of protein.

I saw this product https://www.thomassci.com/scientific-supplies/Tube--o--dialyzer, do you have any products like this that you are more comfortable with? Will it compromise the quality of the purification?

Hi everyone,

I have been working on purifing Tn5 transposase for quite a long time. Basically following the protocol from http://genome.cshlp.org/content/24/12/2033.full. Occasionally I got one batch of transposase having activity, but after around 3 months, activity was not detectable any longer. Other than that batch, I got no more active protein. According to protein gel, the protein size is correct, roughly 53.3 kDa. Is there any tricky step that I missed? Any suggestion or help would be appreciated!

Best!

Hi,

I've been routinely using Dialysis for removing salts after we do protein purification using Ni-NTA or any other chromatography. But I'm curious to know how economically and operationally feasible this process is when we move to Industrial set ups?

Since we have lower volumes (15-20mL) of protein in R and D lab it's easier to perform in beakers, how would it affect the process when we move to 15-20L of the same protein?

I'll be happy to read if anyone has any literature in this area.

Thanks

Hello,

I expressed a protein with a his tag (65 kDa) and purified it by Ni-NTA affinity chromatography. The amount of protein that I got was huge, so I didn't wonder about the aggregation in the fraction tubes overnight. The next day I spun them down and made an SDS PAGE. I repeated the SDS PAGE several times with different amounts of protein to make sure I was doing it the correct way. Everytime I get an additional band (and smear) right above my protein (the thickest band is my protein). I then diluted my protein 1:10 and purified it again by Ni-NTA affinity, but I got exactly the same result.

This puzzles me a lot, because my colleague expressed this protein many times and purified it the same way, and he never got this additional band. So now I am wondering, what could be the reason for the sudden appearance of the band?

Could it be somehow that the amount of protein is too much for the column? Was the expression too long (around 16h at 16°C)? I am not sure how to proceed now. Repeat the expression (but what should I change) or rather purify it further, e.g. with IEX?