Hi,  after sonication the lysate would be turbid and more viscous as the cell contents are released.  Resuspend the cells in cold buffer and sonicate not more than 10 sec per cycle and pause for 30 sec.  During sonication, keep the cells in ice bucket.  Ensure, your wash buffer is free of protease and not contaminated. 

Hope, you had sequenced your recombinant plasmid to ensure that the codon is in proper reading frame. 

I haven't noticed the lysate becoming viscous.. the plasmid is fine. I got it from addgene and performed a digestion after midiprep which gave correct bands. How much time is generally necessary to complete lysis of E.coli using sonication?  

Hi Amila

I would like to point the following things:

1.The sonication power-I think you can increase the power to 100 Watts 

2.In the wash buffer, I can add other agents which aid in cell lysis depending on your protein of interest like lysozyme, triton X 100,NP-40 NaCl,Sucrose,etc.This you need to optimize for your particular protein.

3.You need to check whether your protein is coming in the pellet or the supernatent after centrifugation. If your protein is forming pellet, it might be getting into inclusion body, rather than soluble fraction

4.Have you checked the SDS-PAGE of uninduced and induced cells?

5.Do yoru run the unbound and wash fractions as well in the SDS-PAGE after Ni-NTA chromatography? because it may be that your protein isnt binding to the resin. 

6.For optimum binding to Ni-NTA resin you need to check the pH of the buffers and there should not be any EDTA in any of the buffers.

Hope this helps

Good luck :) 

Hi Amila,

your problem definitely in the cell lysis by sonication. As Shreyasi suggests, you need to increase the power of the sonication. I use near the maximum power of the sonicator with 4 cycles each cycle 30 sec sonication and 30 sec pauses on ice. After good sonication the solution will be warmer. After sonication there will be pellet because you have big biomass of the cells.

You can also try alternative methods for cell lysis, e.g. you can freeze the cell suspension (no matter what buffer you use to resuspend cells) at -20C and after thaw you will see how viscose will be the suspension. Consider to use DNAseI (and buffer for protein lysate preparation compatible with its activity) to decrease viscosity and improve purification of your protein on Ni-NTA.

By the way, E.coli has proteins that can non-specifically bind to the Ni-NTA resin, so if you have good protein lysate there will be always some protein contaminats in your eluate. You might need to check the presence of your protein.

Next time, take an aliquot at each step of the purification and run on SDS-PAGE (and coomassie blue staining) to know where things gone wrong.

We routinely do sonication 4 X 30sec on ice and measure absorbance (dilute the cell suspension in LB 1:50 or 100) at 600nm before and after each cycle of sonication to make sure the sonication worked (we usually get 90% reduction in the absorbance after 3-4 cycles of sonication).

https://www.diagenode.com/files/protocols/Bacterial_Cell_Disruption_with_Bioruptor_protocol.pdf

Do gel extraction after sonication instead of column extraction. You will not  get contamination with other proteins also. i did sonication at 60 amplitude,  "Lysed bacterial cells in 1X PBS were subjected to sonication at amplitude of 60, 0.6 cycles for 5 x 30 second bursts with 30 seconds cooling period between each burst on ice until the bacterial culture became clear"

for bactrial pellet from 1 L culture, you need to resuspend into 40 ml of lysis buffer (EDTA/DTT free) vortex it for a while to dissolve completely. then incubate on ice for 30 minutes. then perform the sonication with 3 seconds OFF/ and 5 seconds ON, for 5-10 minutes with 38 % amplitute. then spin down at 14000 rpm for 45 minutes. this will help you to get complete cell lyisis and your protein more in soluble fraction. then go for purification. 

good luck 

I took the IPTG induced overnight culture of 1 liter, centrifuge at 12000 rpm for 10 minutes. Dissolve the pellet in 1x PBS buffer. Sonicate it at an amplitude of 60 and 0.6 cycles for 30 sec . 30 seconds cooling. Repeat the process of cooling and sonication till the solution becomes clear which indicates lysis of the bacterial solution. Centrifuge the sonicated sample again and dissolve the pellet in 10-15 ml and run the sample you got in the last sample in Biorad Maxi Protean gel. Cut the desired band . Repeat the gel running for 4-5 times and reextract the protein from gel with 5% SDS., followed by destaining of the extracted protein with equal volumes of 1%ammonium bicarbonate and methanol. Then perform dialysis against MilliQwater. Finally get the concentrated purified sample of 1.5-2.0 ml of protein. This gives purified protein without  background of other proteins when obtained by column based purification. Location of protein also depends on the vector we used for cloning. Sometimes, we get the protein in soluble form, sometimes in insoluble form (Protein). So, you need to check where the protein is lying after sonication. Whether its in insoluble form or soluble form after centrifugation of the sonicated sample

Thanks for all the good suggestions, I'll do another experiment soon and I'll let you know how it turns out. 

Do not conclude based on enzyme digestion of plasmid alone.  PCR amplification of insert CAN generate mutation although your Taq enzyme is fool-proof.  In fact, your template purity matters when amplifying the insert and error can arise if it is not pure.  Hence, first check the insert sequence/ reading frame.  If, the lysate is not viscous after sonication,  then your cells are not lysed properly. Best is to prepare the lysis buffer again and recheck the SDS concentration.  Use all fresh solution and not to use the old one, including water..

If you have no induced band of the correct Mwt by SDS when compared to uninduced control then it is more than likely an induction problem/sequence problem. If you do have a band and post sonication binding you have no visible protein detected then this could be a number of problems;

1. Insufficient lysis: sonication/ with lysozyme. Check lysis under microscope and post spin protein assay the supernatant. Analyse pellet and supernatant samples by SDS PAGE. If mostly in pellet then more than likely it is in inclusion bodies. You can modify the induction process to try and achieve soluble protein or extract and re-fold from pellet. Heat is usually bad for protein so a cooling step during sonication is a good idea. 

2. Protein does not bind column: Hist tag is hidden (steric hinderance) and therefore unable to bind IMAC so protein remains in supernatant or lost hist tag due to proteolysis etc/over done on sonication (denatured protein)

Depending on what you want to do with the protein you could purify using denaturing conditions on IMAC

3. Concentration of protein is very low and is out competed by host proteins/masked. Try a capture step such as IEX to increase product concentration and remove some host proteins

bw

Ian

 Dear Zuko, 

First of all, I agree with Kumar and Wilkinson. You must be sure about that expression system is correct and expressing the correct protein (size in the SDS-PAGE, PCR analyses, plasmid profile digestion, sequencing, etc.).  one question: are the protein in inclusion bodies or soluble form into the e. coli cells? After, be sure that all cells are lised. to lise cells, you can use lisozyme or a buffer or high pressure (homogeneizer system). Check the relation between cells amount and buffer during the sonication, maybe you are large amount of cells related to buffer and you need increase sonication time/repetitions and beware about temperature. Personally, I do not like sonication and I like to check each step to check where is the protein e if it´s ok by analyzing pellet and supernatant. For purification, check if the protein are present in the solution before passing through the column and if it is in the correct form to link to the Niquel (IMAC) like solubility, hidden tag, dissulfite bonds presents, amount of protein is enough to the volume of resin, Did you check if the protein passed through the column or it remained into the column after elution?  

Dear Amila,

Protein expression also depends on the type of E.coli cells you are using in to which you are introducing your construct. Try to check the sequence of the gene of interest for the presence of rare codons.If there are rare codons in your sequence, then normal BL-21(DE3) E.coli cells will not be able to synthesise full-length translatable protein. While Rosetta gami strains of E.Coli have the tRNA pools for rare codons and thus have the  ability to translate rare codons present in the mRNA of the gene of interest. The presence of rare codons in the gene sequence can be verified by online available tools such as Rare Codon Analyser, Rare Codon Calculator. One research paper shows that while trying to express a 25 KDa coat protein of Citrus Tristeza virus , BL-21(DE3) cells were able to synthesise only 14 KDA truncated protein while Rosetta strains synthesised full-length 25 KDa protein. Due to unavailability of tRNA to supply amino acids pertaining to rare codons, translation is stalled, or premature termination  or misincorporation of amino acids occurs. Also, if you have antibody available against the protein you are trying to express, you can do western and confirm. If you have truncated protein, then there will be 2 bands available in SDS PAGE due to formation of truncated proteins when expressed in BL-21(DE3) while it gives a single band of desired molecular weight incase of Rosetta gami strain .

My apologies for not replying sooner but for those who were interested, sonication worked and I finally have my protein! There were two problems: 1. Sonicator didnt work initially. I hardly had any proteins in my lysate and 2. I switched to Rosetta Gami 2 (DE3) strain which is producing my protein really well. I have also been able to purify my protein (his-tagged) by using IMAC, unfortunately I have some background proteins in my eluate. I created another researchgate thread for this: https://www.researchgate.net/post/How_to_reduce_the_amount_of_coeluates_after_IMAC

Amila Zuko. Thats great news . My suggestion to switch to Rosetta gami strain worked and you are able to express the protein. Yep. The problem is with sonicator. If it works, at the end of sonication, you should be able to see clear lysate after few cycles of sonication. But, if the bacterial solution has no change means lysis of bacterial cells is not happening.