The pellet contains insoluble proteins, some of the membranes, unbroken cells, and cell fragments. If the sonication was insufficient, there will be a lot of unbroken cells and cell fragments. If the sonication was too vigorous, the samples may have overheated, causing protein denaturation and precipitation.

The important thing is how much of the overexpressed protein is in the supernatant.

The pellet contains insoluble proteins, some of the membranes, unbroken cells, and cell fragments. If the sonication was insufficient, there will be a lot of unbroken cells and cell fragments. If the sonication was too vigorous, the samples may have overheated, causing protein denaturation and precipitation.

The important thing is how much of the overexpressed protein is in the supernatant.

When the amount of total protein in soluble fraction is larger than that in insoluble fraction on SDS-PAGE, it is due to insufficient sonication. If you want to completely lyse the E. Coli, you might want to use Lysozyme and TritonX100 treatment before sonication.

Hi Ketul,

It would have been nice if you had shared the gel pics. But to answer you question i will suggest it can be because of two reasons 1. In sufficiant sonication as you suggested or 2.In sufficient heating while sample preperation in this case protein will not be linearized and same protein can come as multiple bands. I will suggest you to run whole cell lysed in SDS loading buffer as a control. Take a pinch of cell pallet with 10 uL tip and add to 20 ul of Loading dying similarly add pallet and soluble fractions in different tube with loading dye. Heat all the samples at 95 C for 10-15 mins. Make sure samples are not dry or u can add more dye and take more samples. Now load on SDS gel. You will be able to get clear dicreat bands and can compare it with whole cell lystae

Insufficient sonication , try lysozyme or suitable detergents like tween series or Triton before sonication.

Regards

Abhishek Sharma

Thanks for these valuable suggestions.

Here is the gel pic. Discription

Lane1 marker

2 control sup.16°c

3 control pellet 16°c

4 .5mM iptg 16°c sup

5 .5mM iptg 16°c pellet

6 .25 mM iptg 16°c sup

7 .25 mM iptg 16°c pellet

8 .125 mM iptg 16°c sup

9 .125 mM iptg 16c pellet

10 .5 mM iptg 20°c sup

11 .5 mM iptg 20°c pellet

12 .25 mM iptg 20°c sup

13 .25 mM iptg 20°c pellet

14 .125 mM iptg 20°csup

15 .125 mM iptg 20°c pellet

Hii ketul

I think your protein is going in inclusion bodies, which can be seen in pellet fractions. You can try another vector, varying concencentraion of inducer, incubation period and temperature(4-40c) to get rid of protein into inclusion bodies. You can also purify the pellet protein and refold it, if you need it in active form.

Hope this works. Best of luck

Vijay

Dear Vijay Singh,

My question was, why I am getting more bands in pellet fraction then soluble?beacuse ideally speaking, pellet fraction should only have membrane protein.

I my case what would be most probable situation,

is there large number of unlysed cells ?

Or sonication cleaved the protein in smaller fragments?

Or protein is not expressed (but I got thick band in pellet after induction at 25, 30, 37 °c )?

Ketul,

If the problem is only of sonication you can reduce the volume of sample during sonication or increase the sonication time. your protein is expressed and sonication does not affect protein integrity so donot worry about it.

please check your protein is in soluble or insoluble fraction of host cell, this is most important thing in my point of view.

vijay

To answer your query "My question was, why I am getting more bands in pellet fraction then soluble?beacuse ideally speaking, pellet fraction should only have membrane protein." I feel your protein is getting expressed nicely but inclusion bodies are forming more so only u are getting it more in pellet. And as other said it may be not linearized properly while sample preparation but I think if You express your protein at lower temperature like at 16 °C in 0.5 M sorbitol containing LB media, it will solve your problem. I have done it for my proteins. Briefly for trial " Escherichia coli cells harboring your construct should be precultured in 5 ml of LB medium having antibiotics overnight at 37 °C. The cells from the preculture then inoculate in 1000 ml of LB medium containing antibiotics, 1mM betaine (optional), and 0.5 M sorbitol. The bacterial cells should be grown at 37 °C to an A600 of 0.6–0.9 and induced with low concentration of IPTG (u can try 0.5 mM) for 12 h at 16 °C. Then pellet and process as you want.

All the best