I think your cells are not sonicated well because at 6000 rpm only intact cells can pellet and that is why there is nothing in the supernatant. To assist sonication try lysozyme lysis followed by sonication and centrifugation at 25000 g for 30 min. Also if not done try to reduce the post induction temperature to 18 degree, usually helps.

Is it possible that this protein is aggregating in your lysis buffer? Sounds like your protein is not soluble. I would check literature to see whether others used similar buffers for purifying this protein or other proteins in the same family/fold.

Could you please specify all your procedure, from cell disruption to sds-page?

Dear Dhananjay Gotarkar your desired protein is cytosolic or membrane protein if cytosolic you can use cell lysis buffer and sonication method then high speed centrifugation then collect supernate and pass throuh column and wash with elution buffer then run on SDSPAGE 

Hello Dhananjay,

Whether your protein is in soluble form or pellet form, still you can purify it by using Ni-NTA agarose (Qiagens).

Please explain your procedure in detail? So that we can sort out the problem.

For time being, if you can get your protein in bacterial cell debries, simply dissolve it in 8 M urea and then try to purify your protein using Ni-NTA agarose under denaturing conditions.

#Manoj Pohare.

Glad to see your answer. How are you?

Have you used Ni-NTA coulum to purify protein from cell debris (pellet)? let me know. that will be helpful.

thanks

Hi Dhananjay

You can try 3 freeze -thaw cycles after harvesting cell pellet then resuspend the pellet in chilled lysis buffer(5-10mL for pellet from 500mL culture) and add lysozyme (10mg/mL) incubate on ice for 30min with periodic flicking on ice bath. Sonication may degrade the protein so avoid sonication or use very small burst on ice bath (2s burst with 5s interval). Centrifuge lysate at 14000xg for 10min @ 4C. I hope you will have the protein in your supernatant. If you have Ni-NTA column then use it otherwise you can just add Ni-NTA slurry into lysate and keep it on roller mixer in cold room or in ice bath on rocker for 2-3 hours. discard the lysate after centrifugation while treat the beads for protein elution with same buffer with higher concentration of imidazole. I hope this works. you also need further purification steps. 

Feel free to ask if further details required

Try refolding using on-column procedures. Your his tag should be 6x at least.

Procedures are in the literature. For starters, you may try mine from prion work.

Hello Dhananjay,

I am fine. I never tried it for cell debris, but I tried purifying proteins by Ni NTA agarose under denatauring conditions like 8 M Urea. If you want to opt for it, solubilize your debris in 8 M urea and then follow Ni NTA purification under denaturing conditions.

If you need more help in this regard contact me on [email protected]

Hello Manoj

thanks for the advice. will look into it and let you know.

Dear, Mangal Sing, Ozgun Erdogan,  Omar Gonzalez-Ortega, Arvind Singh Chandel , Manoj Pohare, Muhammad Faraz Anwar, Wieslaw Swietnicki.

Thank you all for your valuable answers. Let me explain my procedure in brief to make it easy to get specific answers and solve the problem -

1) I started with inducing the protein followed by spinning down the cells. to that added 1X PBS and sonicated for 8 cycles of 45 seconds, then I spin at 6000 rpm for 5 min. saved the PELLET and respin the supernatent at 12000 rpm for 20 minutes. separeted the SUPERNATENT and INCLUSION BODIES. to all three (pellet, supernatent and inclusion bodies) mixed with 6X SDS buffer followed by heat denaturation 98 degree Celsius for 8 minutes. and run on  10%SDS page followed by coomassie staining.

2) In addition to (1) used Lysozyme before sonication.

3) In addition to (1) tried freeze thawing using -80 degree Celsius

Result (1), (2), (3)  - I could not see any expected size band of my protein in SUPERNATENT and INCLUSION BODIES, but I can see my induced protein in PELLET. (all compared to control, uninduced samples.)

Note -

PELLET = cell debris etc settled down after centrifugation at 6000 rpm for 5 minuted after sonication.

SUPERNATANT = upper layer obtained after centrifugation at 12000 rpm for 20 minutes.

INCLUSION BODIES = pellet after centrifugation at 12000 rpm for 20 minutes.

I hope this will help to understand the problem I am facing in getting the protein available for purification. I will use the Ni-Sepharose matrix to purify my HIs-tagged protein further.

Will appreciate your answers.

thanks

From your explanation, it seems that either your inclusion bodies are precipitating with the cell debris or your sonication is not enough for rupturing the cells.

Why do you precipitate the cell debris separately?

I generally follow this protocol.

Induction (take out control), collect the cells, sonication in the desired buffer, separation of  supernatent and pellet at 15000 rpm for 10 min. Pellet will be the mixture of cell debris and inclusion bodies (if your protein is expressing as inclusion bodies). Wash the pellet thrice with distilled water and finally with same buffer used for sonication to remove the cell debris. In this way you will get the inclusion bodies as white precipitate.

please check it in the way explained above.

Possible causes and solutions are:

• Sonication may be insufficient: Cell disruption may be checked by

microscopic examination or monitored by measuring the release of

nucleic acids at A260. Addition of lysozyme (up to 0.1 volume of a 10 mg/

ml lysozyme solution in 25 mM Tris-HCl, pH 8.0) prior to sonication may

improve results. Avoid frothing and overheating as this may denature

the target protein. Over-sonication can also lead to copurification of host

proteins with the target protein.

• The protein may be insoluble (inclusion bodies): The protein can usually

be solubilized (and unfolded) from inclusion bodies using common

denaturants such as 4–6 M Gua-HCl, 4–8 M urea, or strong detergents.

Prepare buffers containing 20 mM sodium phosphate, 8 M urea,

or 6 M Gua-HCl, and suitable imidazole concentrations, pH 7.4–7.6.

Buffers with urea should also include 500 mM NaCl. Use these buffers

for sample preparation, as binding buffer and as elution buffer. For

sample preparation and binding buffer, use 10–20 mM imidazole or

the concentration selected during optimization trials (including urea or

Gua-HCl). To minimize dilution of the sample, solid urea or Gua-HCl can be

added

Hope it be useful to you! good luck!

Hello Dhananjay Gotarkar.

I'm having the same problem. I am using E. coli Shuffle T7 strain for my recombinant protein induction. I made a pilot expression and I saw my protein in the soluble fraction. After the induction and lysis (using bead-beater or sonication) of 1L I checked the recombinant protein by sds-page and I saw it in bacterial cell debris. I've tried using lysozyme and Triton X100, but it still in the cell debris. Did you find a way to solve your problem?

I will appreciate your answer

Thanks

Hola Jessica

Yes, I was able to purify this protein.

I did freeze-thaw the pellet at -80 degree, followed by rigorous sonication and storing in ST buffer overnight, followed by diluting with 1% Sarkosyl, 2% Triton X-100 and 20 mM CHAPS for better solubilization of the protein before sample injection into the column.

Let me know if you need more details. Good luck

Thank you for your answer Dhananjay.

I'm going to try what you told me and hope for the best.

Thanks again