If you want to make quick extracts from whole cells, I would suggest to take 1ml of culture at OD(600)=2.0 (or more or less depending on the density of your culture), spin down the cells and resuspend the pellet in 50 µl of 1xSDS loading buffer. Boil for 10 min, then spin at max. speed for 10 min. The sample will be viscous, but if you take 8 µl from the very top of the liquid, you should easily be able to pipet it.

I always use this protocol to look at the total lysate and I never have problems with it. If you stick to these values for the OD and for the volumes, then you should get a nice Coomassie-stain, and Western blot should also work perfectly...

I hope that helps....

Are you removing the e coli cell wall with lysozyme before putting them into loading buffer?.

No, do you think it would be required? I assume the cells are ruptured anyway when boiled with the loading buffer?

The samples I have run so far are from frozen whole cells from a grow-up I did a few weeks ago. I have a feeling this could also not help matters, and shall run a fresh batch next time.

If you want to make quick extracts from whole cells, I would suggest to take 1ml of culture at OD(600)=2.0 (or more or less depending on the density of your culture), spin down the cells and resuspend the pellet in 50 µl of 1xSDS loading buffer. Boil for 10 min, then spin at max. speed for 10 min. The sample will be viscous, but if you take 8 µl from the very top of the liquid, you should easily be able to pipet it.

I always use this protocol to look at the total lysate and I never have problems with it. If you stick to these values for the OD and for the volumes, then you should get a nice Coomassie-stain, and Western blot should also work perfectly...

I hope that helps....

Hi. Lysozyme is not required, but it could increase the extraction of soluble proteins from the lysate.

http://www.piercenet.com/browse.cfm?fldID=3C6EACCB-232E-44DC-913F-6D9B16BEF8D5

Adding DNAse to the extract could also help reduce the lovely gelatinous lump (which I usually call snot:)

Hi, did u try to sonicate this gelatinous lump?

I had this problem too ...... I ended up lysing with B-PER or bug buster with some DNAase added.

Micheals suggestion are quite fine.

You should think about something in the background:

1. You need to estimate how much protein is in your lysate. OD280 nm gives you some idea. Absorbance 1 = 1 mg/mL.

2. Now you have to think about the composition of your proteins. Bacteria have only a small amount of proteins with a high molecular weight. To get an overview, a gradient gel is helpful (4 - 10% ... 15%). You should have some idea about the MW of your protein of interest. It's should end somewhere in the middle of the gel.

3. The staining method used is important for the amount of protein you will load on your gel. A whole cell lysate can be added with about 20 - 100 µg/lane. It's will be sufficient for a Coomassie stain. For a silver stain 1 - 20 µg/lane will be fine.

4. If you are planning a western blot to identify one (or more) of your proteins, 5 µg/lane should fit your requirements.

5. Western blots are often fine, when you are running a single wide lane only (+ a single slot for the MW marker). After the transfer you go first for a staining with Ponceau S (it's removable). Here you can see how to cut the membrane into 2 - 4 mm strips. Each of them can be stained with different methods. There are methods for proteins, glycoproteins, several gold and silver staining methods, ink staining's, and may be several specific antibodies ...

and now: Adjust the protein concentration according to your needs and dilute the sample in your SDS and mercaptoethanol-containing sample buffer.Glycerol will increase the density of your sample. So you can apply the sample much easier into your slots. Saccharose can be used as well without diluting you sample.

I agree with Michael, just spin down 1 ml of overnight culture of bacteria and resuspend in 20µl of loading buffer with SDS PAGE. Boil it for 10 minutes, centrifuge quickly and load the supernatant on the gel. I always use this protocol, and it works well, even for western blot.

In a past lab we would use a 25 gauge needle and 1ml syringe to sheer the DNA and eliminate the viscousity. suck up and eject the extract about 5 times eing careful not to get bubbles. admitedly this was not on E.coli but on A549 cell but it worked to get rid of the visous blob.

Hi Siobhan

As per my experience i am putting here a procedure how i perform SDS-PAGE on cell lysate. Hope it will be helpful for you. If you try this and find it useful please let me know.

Perform cell lysis of the cell pellets obtained from 500 µl culture with 50 µl of lysis buffer (50 mM potassium phosphate pH 7.8, 400 mM NaCl, 100 mM KCL, 10% v/v glycerol, 0.5% v/v Triton X-100, 10 mM imidazole). In your case you can approximate the amount of cell pellet that would be obtained from 500 µl culture. Make a good suspension by vortexing. A cloudy appearance is the indication of complete suspension. Heat the suspension at 50°C for 5 min. Take an aliquot of 15 µl in a 1.5 ml screw cap tube, add 3 µl of reducing sample buffer (60% v/v glycerol, 300 mM Tris-HCI pH 6.8, 12% w/v SDS, 600 mM β-mercaptoethanol, 0.6% bromophenol blue), vortex briefly, spin briefly, load onto the tube raft and boil for 5 min in a water boiler. Spin briefly. Load the reduced sample (18 µl per well) on 12% SDS-PAGE gel (either lab prepared or commercially available precast gel). Run the gel at 120V for 60 min. Stain the gel with 0.2% Coomassie blue overnight with gentle shaking on an orbital shaker and wash with MQ water several times until the back ground is clear.

The protein band should be visible in about 3 hours of Coomassie blue staining but I prefer overnight for staining all bands that have lower concentration of proteins.

DNAce should work to get rid of the clump. But If your dimers are not cross linked, SDS PAGE would give you only the monomer population.

I would like to give you a simple protocol:

1 ml of culture medium (at the time you want to study) at OD(600)=1.2

pelltet is resuspend in 100 ul dH2O or lysis buffer

Add 25ul 5X Sample buffer

heating and centriguge => 5ul sample per well

GOOD LUCK TO YOU

I am working well with this protocol

u just take 1.5 to 2 ml of overnight grown culture of E coli, in a centrifuge tube and pellet the culture. Discard the sup, in pellet fraction add 50 microlitre of SDS gel loading dye , mix with help of pipette and boil it for 15 minutes. Load 10-15 microliter of this sample i a well.

I use 300ul of 1xSDS loading buffer per ml of pelleted cells with OD=0.6 and boil (adjust depending on OD). Works great and sample can be pipetted no problem.

you take culture in 1.5 ml eppendorf then centrifuge it 8000 rpm for 3 mint then descard the supernetent and mix the pellet in 200 ul autoclave distiller water after resuspend add 50 ul 5 X SDS -PAGE dye and mix then put in boiled water for 4-5 mint. then lade in SDS-PAGE . nad run. Its my daily routine work and its really .easy

This is a common problem you find when u deal with recombinant membrane proteins. Actually there are two approaches you can take First do protein fraction of membrane protein using ultra centrifuges and second change vector so that the periplasmic protein of yours can be expressed in cytosol. I can suggest you can change vector to pBAD TOPO expression vector from Invitrogen. Initially I had also hard days expressing membrane proteins, changing vector solved my problem. I am now getting good blots of 12 transmembrane proteins.

Thank you everyone, I resuspended whole cells directly in loading buffer and boiled, as most of your have suggested. They still go rather 'gloopy' but when I spin and take the thinnest part of the supernatant it's ok.

I also used fresh samples this time instead of from frozen which seemed to help. The whole cells shall be run against my protein fractions.

I shall see how the westerns turn out tomorrow....

Plenty of good suggestions are already listed here if you only need run SDS-PAGE of whole cell samples. But if you try to "compare distribution of monomer:dimer populations of a recombinant protein expressed in E.coli", SDS-PAGE may dissociate protein dimer, thereby failing to give your real distribution of monomer:dimer population. In this case, you might need use Blue Native PAGE ( a very good paper about BN PAGE as showed: Analysis of membrane protein complexes by blue native page. Practical Proteomics, 1-2/2006). Good luck.

You may try to sonicate to breakup the DNA or easier still, just add some extra glycerol, that should keep the sample from floating away.

Like Michael Taschner, I pellet the cells from 1 mL of induced culture but I resuspend the cells in 100 mL of 3x loading buffer. I mix several times with a pipet and then boil for 5 minutes. After that, I dilute 3x with water and load the equivalent of 50 µL of culture: in this case: 5 µL of boiled sample + 10 µL of water and it is not too visquous,

good luck,

Colette

sorry, of course I resuspend the cells in 100 µL and not mL

You can pellet 1 ml of culture and resuspend in 50 ul of 1X PBS and 50 ul of 2X Laemmli lysis buffer and boil the cells for 20 min (u can observe that the turbid suspension would have turned transparent or semi). Then spin the tubes at max. speed for 2-3 min. Collect the supernatant for loading into the gel. We regularly practice this in our lab during protein expression studies.

well, in my experience it is not good idea to add directly pellet with your Laemmli sample buffer (LSB). Better to resuspens the pellet (after removing the medium and washing with buffer) of pre-french pressed sample with 300-500 uL, depending on concentration of your cells. Then take 50 ul of the resuspensed cells and add LSB prior to boiling. Optional, you may add urea on your LSB to make sure all proteins in the cells are denature. I would kept it for 5-10 min stands at RT before boiling when you include urea on your LSB. Good luck.

In my experience, adding DNAse prior to boiling helps to get rid of the snotty stuff (DNA?) and ensures that the samples do not float out of the wells during loading.

equal volume of 2x SDS loading buffer and cells, then sonicate, boil, load 10ul.

The gelatinous blob is probably DNA, you could do as Nicolas Berry suggests, and in addition you could add urea to your sample buffer, and when you have boiled your samples do not put them on ice, just load them and it should be fine. It works for me!

However, when you talk about the distribution of monomer/dimer forms of your protein, are they covalently linked or fusion proteins?, as western blotting might not be the way to go if they are not covalently linked.

I agree with Andrew, your problem is the genomic DNA. To eliminate the DNA just sonicate samples at 5 microns of amplitude for 10 s. in a MSE ultrasonic disintegrator with an exponential probe. Put the sample tube in a small beaker with ice to prevent overheating the sample. After sonicating you can boil and load the sample onto a SDS-PAGE gel and proceed with your protocol

High levels of viscosity in whole cell lysates is, for the most part due to the presence of the DNA released on cell lysis. There are several ways to deal with this, since your experiments seem to require only the protein of interest. You can use DNases to get rid of the DNA, however, for practical purposes, this may prove to be too expensive since the amount of DNAse required to get rid of DNA from a cell lysate will be very high.

Consequently, you can use a sonicator to breakup the DNA into smaller fragments, followed by centrifugation at high speeds for 20-30 minutes, to get rid of the DNA in the form of a pellet. The supernatent should have a water-like consistency, and no viscosity.

Hi,

It sounds like you have lots of good advice already. I just wanted to add that I wouldn't be so concerned about French pressing your cells in the first place. French pressing (2x 10,000 psi) followed by sonication was a routine part of many of my purification procedures and I never experienced protein shearing.

I do the same, but with a few changes...

Pellet cells from 1 ml confluent culture and re-suspend in 70 ul of Water (or any buffer, say 1 X PBS) + 30 ul of 10M UREA adn 20 ul of 6X loading dye that has SDS and beta-ME..

Boil at 94 C for 15 min. Quick Spin and load 15 ul for a 10 well comb or 10 ul for a 15 well comb.

I hope I am clear..

UREA is essential when making whole Cell lysates...

Santosh.

Please clarify.. As I understand, you are interested to study the oligomerization of a protein produced in E. coli.

Are you using SDS-PAGE just to check the protein production or to check the oligomerization?

You can as well use native-PAGE (make sure your protein pI is lower than 7.0) followed by western blotting to check the oligomerization..

On a comprehensive scale u can do gel-filteration and DLS, to identify oligomeric status of the proteins..

Good luck.

Santosh.

So, the main reason whole cell lysate is that viscous is due to DNA. 

Use a DNAse! What I do is this:

Take 1ml of whole cell lysate, spin down, discard supernatant. Re-suspend in your buffer of choice, add 1uL of Benzonase, and vortex a bit. Then add in your sample/loading buffer, and boil. You'll have no trouble loading the sample at this point.

I use Benzonase when loading samples that have pre-lysed cells, as well as my sonicate. 

i've done quick spin down of pellet, boil in loading buffer, then do a quick sonication (break up DNA).  A few aggressive pipette up/down can sometimes break it up enough too.  DNAse may also work.