Protein Expression and Purification Troubleshooting Forum

More than one protein induced

Wed, 18 Nov 2009 02:50:55 -0800

Hi!

I have a problem with an his tagged protein. I use the T7 expression system in e coli with a pET vector. The problem is that after induction I see 3 fat bands which are induced. This tag is C terminal. When I try purification I purify only the smallest of the 3 proteins. Are this really 3 proteins or is there another possibility? How can I purify the right protein?

Thank you for your help

Protein aggregates

Tue, 17 Nov 2009 11:48:49 -0800

Hi everybody. After purification of a membrane fraction, I have measured the absorbance at 215nm (peptide bond absorption) and at 280nm (absorption by aromatic amino acids (tyrosine, tryptophan and phenylalanine)). Now, at 215 nm the peak corresponding to the monomer is sharper than at 280 and the peak corresponding to the aggregates almost disappears. THe preparation os identical in both measurements. Any hint on why this is happening? And which measurement at this point is most reliable? Thanks a lot!

water withdrawal with PEG20.000

Fri, 13 Nov 2009 03:15:30 -0800

Hi all

I used PEG20.000 to reduce my protein sample volume from 100μl to 50μl. I put flakes of PEG20.000 on a microdialysis tube. Before the water withdrawal my protein sample contained 20% glycerol. So my question is: did PEG20.000 drag glycerol along with the water?. Does my 50μl protein sample contain 20% or 40% glycerol??.

Please help a biologist who does not know much about chemistry. I would be very gratefull for an answer!

Troubleshooting GST purification

Thu, 05 Nov 2009 10:54:26 -0800

I'm trying to purify a GST tagged protein. We see expression in the lysate, sonicate, but when we incubate with GST beads, we lose everything. We see non specific proteins in the washes, but by elution time no proteins at all are left. We are incubating ON at 4 C, without protease inhibitors. However, we should not see such complete protein degradation. Any suggestions about where we are losing the protein?

Thanks!

Amount of inoculum?

Tue, 03 Nov 2009 06:41:29 -0800

General practice for recombinant protein expression in E. coli is
1. Transform the cells with the recombinant plasmid and spread on LB agar plate
2. Pick up a colony in eg. 5 ml LB. Grow for few hrs
3. Use part of this culture eg. 0.5 ml for culture initiation in 100 ml LB.
4. And then go ahead till the desired OD achieved.
My doubt is for the step 2 and 3. Why are the cells initially grown in smaller amount of media? And then only part of the grown culture transferred to larger volume of media? Why not to grow the colony directly in 100 ml LB? Or why not to use whole culture obtained after step 2 for step 3?
Has anyone ever got different/negative results after doing so?

BL21A or simliar

Thu, 29 Oct 2009 13:48:04 -0700

My lab has recently started going through a lot of DpnI and I have found a source for the plasmid as well as a method of purification. However, the enzyme needs to be produced in a dam- cell line. The only IPTG inducable cell line I have found seems to be BL21A (DE3) but it was suggested by someone on another site that the person who wrote the paper/used the cell line also created the cell line. I have tried locating the author however the article is over 20 years old and I quickly hit a dead end. I have not been able to find it or an equivalent anywhere. I can find plenty of plasmid propagation cells that are dam- (INV110 SCS110 JM110 GM33) but I cannot seem to find a dam- protein expression line.

Does anyone know where I might be able to acquire BL21A (DE3) or an equivalent? Or can anyone suggest something else entirely?

Thanks so much!

Role of imidazole in lysis buffer

Tue, 27 Oct 2009 23:45:05 -0700

Is there any specific role of imidazole in the lysis buffer used for recovering recombinant his-tag protein from E. coli?

protein expression (temperature issue)

Sun, 25 Oct 2009 07:55:59 -0700

Hi,

I am trying to express a protein from pGEX-5X-1 vector. It was found to be in the inclusion body when expressed at 37C and 30C. I would like to express the protein in the soluble form by lowering the temperature. I am thinking of trying the expression at 18C.

I see that usually papers would induce around 18 hours. I would like to know if 5 hours would be enough to produce the protein?
Any suggestions?

Thanks in advance.

radiolabeling and co-IP with invitrogen dynabeads

Sat, 24 Oct 2009 14:32:59 -0700

Hi everyone,

I am currently working with a radioactive labelled antigen and antibody (with His-tag) against it. I try to do an co-IP with my antigen + antibody and invitrogen Talon (cobalt) dynabeads. I did a control without the antibody and then my radioactive antigen doesn't bind to my beads. So they stay radioactive free. I tried elution with a 500 mM imidazole buffer pH 8 but the elution isn't 100 %. So my beads stay still radioactive bound. Other elution imethod with a low pH 4,5 acetate buffer wasn't better. I want to reuse my beads so I tried to remove the my antibody (and so the radioactivity) with :

- Stripping the beads once with 200 mM EDTA pH 7 and regenerating the beads with Co.
- Stripping the beads two, three and four times before regenerating them.
- Boiling in 2 % SDS, then stripping and regenerating them.

But my antibody is still bound to it with the radioactive antigen.

Does anyone off you have experience with other methods how I can remove my antibody from the beads and so making them radioactive free again.

Perhaps try with a NaOH solution ?

Thanks in advance

A desperate researcher

how to add NLS to the protein

Fri, 23 Oct 2009 02:10:05 -0700

Hello everyone,
I am willing to modify my protein to accumulate it in the nucleus. Can anyone give me a link to the good review on the subject? I'm also interested in more or less detailed protocol for NLS incorporation into the coding sequence if any exist? Thank you in advance!!

Can GST-tag be separated from GST-fusion protein

Sun, 18 Oct 2009 23:53:55 -0700

Dear all,

We need to express and purify a protein with some kind of tag. The most convenient one to us is GST. However, we always get 2 main bands after the affinity column - the GST-fused protein and the GST itself. Since we don't want to cut the tag out this time, we need to purify the GST-fused protein from the GST tag and cannot use the affinity column again.

I know GST will form dimer. Don't know if GST will form dimer with GST-fused protein too? If so, it will be very hard to purify my GST-fused protein from GST.

Anyone has encouter this situation before?

Thanks.

Nest

Protein analog

Sun, 18 Oct 2009 20:53:07 -0700

Hi guys,

Does anyone knows the definition and differences between protein isomer and protein analog?

Thanks for your time

Anasyida

Help with T7RNAP purification

Fri, 16 Oct 2009 05:18:50 -0700

Please note, I have posted this in two discussion groups, so please forgive me for any overlap, I am just hoping desperately for a response

Hi everyone,

Could someone please reference or better yet describe any suitable purification strategies of the T7 RNA polymerase, for its eventual use in a cell free system
I would very much like to know what method has been employed for this enzyme which allows for the maintenance of both enzyme activty and achieving high purity.

Your help would be much apprieciated.

I am currently attempting to purify this enzyme according to Schwarz et al. 2007 Preparative scale expression of membrane proteins in Escherichia coli-based continuous exchange cell-free systems. However during my attempts, I have not been able to achieve an active protein, or at least one that has shown specific activity.

Has anyone figured out a way or any tricks to maintain the activity whilst getting a high quality product?

Thanks

Pat

Protein Extraction from blood/buffy coat

Thu, 15 Oct 2009 03:40:17 -0700

Has anyone tried to extract protein from fresh or frozen buffy coats? I've seen plenty of recommendations for extracting GTP but I'm after the white cell protein. I'd rather extract from buffy coat than blood but any advice is gratefully received!

Thanks in Advance,

Ben

membrane protein extraction buffer

Wed, 14 Oct 2009 06:23:17 -0700

Hi!
Could anybody give me an advice which lysis buffer to use for membrane protein extraction from culture cells. I want to do protein electrophoresis (with denaturated proteins) and I have expressed my protein in COS cells, the protein itself is ATP/ATP carrier, which is transmembrane mitochondrial protein. I have looked for several protocols and I am not quite sure which method would be the best. I have read that some people suggest using the RIPA buffer, but at the same time some people say that its not good for membrane proteins.
Thank you very much in advance,
Minna

Truncated or isomer protein

Mon, 12 Oct 2009 20:55:17 -0700

Hi there,

My protein is 30 kD size with pI is 4.85 determined by in silico. I rarely get single band for my protein with SDS-PAGE, as usually there are two other bands below 30 kD. But only one band appears with anti-His Western blot. When I run the OFFGEL analysis, I got three different bands with pI ranging from 4.5 to 5.0 (this could translate why there are always three bands for my protein). Does anyone know what is actually the other two proteins, is it the truncated form or the isomer of my protein?

Thanks

Expression in yeast

Wed, 07 Oct 2009 00:54:35 -0700

Hello !!
I want to express a viral gene in Pichia pastoris but, this is my first time......any of you have experienece in this issue?, i would like to know if saccharomyces is more convenient or in the case of Pichia which plasmid is the best one. I have read something about pPICZ, pPIC6, pGAPZ. Apparently all of them are terrific (for Invitrogen) but...there are always unofficial hints that can be obtainded from expert hands. If you have any suggestion about a different expresion plasmid for Pichia, please let me know, i don�t want to make the blunder of the beginer.

Thank you in advance!!!!

What about the Baculovirus expression system?

Tue, 06 Oct 2009 03:54:30 -0700

Hello everyone!

I was wondering how to go about expressing some full-length proteins that I will need active. I read somewhere that an 6xHis-MBP tag might be good. Some people in lab have GST or 6xHis and express in E.Coli DE3 pLys. I have no experience with this but a cleavable 6xHis-MBP recombinant protein sounds good. Uhm........ perhaps someone more experienced might have other suggestions I could look into?

All I know is I need the proteins active and I might have to to introduce mutations to compare activity at some point.

Thank you and have a nice day!

Ni-NTA beads and dialysis tubing

Mon, 05 Oct 2009 11:11:50 -0700

Hi there,

I have a secreted protein (95kDa) with a His tag, which I purify by batch-binding the (filter-cleared) culture supernatant to nickel beads. I let the batch-binding take place overnight, then leave the mixture still for a couple hours to allow the beads to settle. Pouring the media/beads tends to take quite awhile because the column clogs up to the extent of blocking all flow. These days, I usually 'resuspend' the clog, though this tends to bash up the beads a bit even if I do it gently. The beads seem to stick to centrifuge tubes so spinning down (even slowly) causes me to lose a lot of yield.

I was hoping that I could speed things up by putting my nickel beads into dialysis tubing (higher MWCO than my protein, say 300 MWCO) so that I don't have to pour and resuspend and wait (repeating ad nauseum for a 6L growth).

So my question: has anyone ever tried this? Will the Ni-NTA agarose beads stick to the dialysis tubing (which would be problematic for purification and re-use)? Does anyone foresee any other complications here?

Thanks in advance for the help,
megan
multanova@gmail.com

Concentrating Protein with centrifuge concentrator but 50% glycerol!!?

Thu, 01 Oct 2009 07:21:00 -0700

I need to increase the concentration of my protein by a 1/2 but problem is - it's in a buffer with 50% glycerol. I'm using the Amicon centrifuge devices and I knew it was going to be slow. After the first 25 min spin I got a sad <1mL in the flowthrough but after the 2nd 25min spin...doesn't look like a drop went through. Is this actually a feasible way to concentrate this protein?......