Protein and Proteomics Method Forum

Q about the housekeeping gene

Wed, 18 Nov 2009 05:41:45 -0800

Hi all,
I'm now IBing a large protein(about 500KDa). I want to know if there is a large enough housekeeping control to do the IB on the same gel with the target 500KDa protein. e.g. we can choose alpha-actin(about 100KDa) together when studying the DMD protein(about 500KDa) in the muscle cells. so all the procedures can be complished just on one membrane. But alpha-actin is only expressed in the muscle. So is there another housekeeping control to use when IBing a large protein?

thank you

J

Microplate Bradford assay

Mon, 16 Nov 2009 19:30:25 -0800

Dear all,

I have small quantity of a protein, and I want to do microplate Bradfor dassay, can anybody explain to me how to set up the BSA standard, and the precise amount in every well, and do I need to dilute my sample or not?

Incomplete denaturing with 8M urea?

Fri, 13 Nov 2009 14:31:41 -0800

Does anyone have any experience with proteins that will not denature in 8M urea? It appears I have several ORFs that are expressed but when I lyse with 8M urea they stay with the pellet. I don't need native structure. I purify on a Ni-NTA column. Any suggestions on coaxing them out?

Protein quantification from paraformaldehyde-fixed cells

Fri, 13 Nov 2009 12:47:53 -0800

Hello,

Does anyone know of a protocol to extract and quantify proteins (e.g. by BCA or Bradford assays) from attached, PFA-fixed cells?

Thanks

vic

Out of expected protein size in SDS-PAGE

Fri, 13 Nov 2009 07:10:37 -0800

Hello all. I'm dealing a protein with expected size of about 50kDa. After i knockdown the protein using RNAi technique, i detected the expression of protein using the primary antibody raised in-house.

For the control lane: a sharp band was detected at about 100kDa. No unspecific band was observed.
For the sample lane: No band/extreme thin band was detected at about about 100kDa. No unspecific band was observed.

The sample should be reduced and denatured, because I have boiled them in Laemmli loading buffer. My experiment on other protein did not face this problem.

The band indirectly showed that it was in dimeric form. However, will this be possible after being boiled?

Thanks for any suggestion!

Bradford quantitatn problem - precipitation

Thu, 12 Nov 2009 22:14:28 -0800

Hi,

I extracted my protein using 2%SDS (scraping, sonication). It precipitate out as blue particle when i added into my bio-rad protein assay reagent. It made quatitation impossible. I never encounter it when i use RIPA..

I tried to dilute it out. precipitate still occur but in lesser extent.

Any help would be greatly appreciated!

Thanks

Sugar solution had changed colour

Thu, 12 Nov 2009 18:36:01 -0800

I had prepared 1M D-galactose and 1M D-mannose solutions few months ago. However it seems to turn "orange".
although I haven't plate it yet to determine contamination, but from the solution itself it is clear and no cloudy effect.

My question is:

1) is this normal?
2) can the solution still be use to elute proteins?
3) what is the possible reason for it to turn "orange"

Thank you.

P/s: I'm sorry cos i not a biochemistry background.

cAMP assay kit

Thu, 12 Nov 2009 12:55:22 -0800

Does anyone who is familiar with using the available kits have a suggestion for a relatively easy-to-use non-radioactive cAMP assay kit?

storing WB blots in PBS-tween

Thu, 12 Nov 2009 07:28:46 -0800

I'm running gel on my proteins total lysates and then transferring to nitrocellulose membrane. I 've heard that you can store the blots in PBS tween for few days if necessary. I've noticed when taking pictures the total absence of any bands for all the preoten antibodies that im using. Is it either because of storing in PBS-T for several days or because of my multiple freezing thawing cycles for the lysates that could change protein conformation?

BSA content in Fetal Bovine Serum?

Wed, 11 Nov 2009 13:35:32 -0800

I know serum contents vary a lot but does anybody know about an estimate of BSA content in FBS or can direct me to a source?

simple pipetting problem

Tue, 10 Nov 2009 20:27:11 -0800

I feel kind of ridiculous posting this, but when I try pipetting Sepharose beads after washing and spinning them...well, I can't. They don't pipette up. I've cut the tips. Do I leave some of the wash buffer in there or what? I'm just using these beads initially to cut down on nonspecific binding. I don't have any problems when I need to pipette PGS beads. Suggestions? (Sorry if this seems pretty elementary..)

Storage of overexpressed protein

Tue, 10 Nov 2009 18:08:31 -0800

Hi everybody,

Im working with protein purification and for that purpose ive stored my overexpressed protein (E. coli BL21, host) as cell pellets in -20 freezer for about 4 weeks. Is there any problem in storing overexpressed protein like this. or is storage of overexpressed protein in -70 most essential ?
pls help
thanks

HELP_Bradford and protein problems

Tue, 10 Nov 2009 11:32:48 -0800

Help!!
Purified proteins using CHAPS/Urea method for running 2D-DIGE (compatible buffer).
Need to quantitate using Bradford so final concentration of sample is 50ug/10ul.

HOWEVER, when I diluted my stock sample in water (as per the 2D-DIGE experts advice - they like to quantitate in water) to do the Bradford assay, it didn't appear to solublize well (got a clearish pellet in the bottom of the tube) and when I did the Bradford assay I got a protein concentration reading of say 10 ug/ul.

SO, I tried diluting my stock sample in PBS, and it appeared to dissolve much better, there was no precipitation or pellet in the tube, but I only got 1/2 the concentration (say 5ug/ul) of the EXACT same amount of sample.

I must have a final concentration of 50ug/10ul for the DIGE reaction to work properly. If I base my dilution on the readout from dissolving in the protein in water does this result in a 2-fold OVERestimate? Or if I base my dilution on the readout from dissolving the protien PBS does this result in a 2-fold UNDERestimate?

Do I go with the water or the PBS reading?
The PBS versus water does not affect the standard curve nor the Bradford dye.
It's just what stays soluble in my sample when i try to dilute it for the Bradford assay.

What would you do?
Has anyone encountered this?
Please advise!!

Oh - and if there is TOO MUCH protein in the final sample for DIGE, it will preferentially label the OVERabunant protiens and I won't get good results. If there is TOO LITTLE protein in the final sample for DIGE, the labelling reaction will not proceed well. Therefore, I really need the right range of protein!

precipitation of proteins from stained cells

Mon, 09 Nov 2009 15:03:55 -0800

Does anyone know if it is possible to precipitate proteins after immunohistochemistry. We have an antibody that works well in situ but may not work to precipitate protein from lysates. Is it possible to permeabilze cells, expose to antibody under natural conditions, and then precipitate out the antibody complexes? Standard Co-IP precipitates proteins but not the one we are interested in, however, the antibody works great to identify our protein in fixed cells in culture.

THANKS!!

Recycling coomassie blue

Mon, 09 Nov 2009 13:39:13 -0800

Hi everyone,

I have a simple question, When you stain your SDSpage gel with coomassie blue, do you recycle the solution?
I know we recycle ponceau solution but I have some doubts about the coomassie blue.

thanks

Bromophenol blue vs. "Coomassie G250"

Mon, 09 Nov 2009 08:37:01 -0800

I have a simple question -- We are trying to detect a very small protein, which should be around 18 kDa although some people say it's detected around 14 kDa.

We normally cut off the dyefront after the proteins run through the gel. (Bis-Tris, 10%, using the NuPage system)

Does it really make a difference to change from using bromophenol blue in the sample buffer to using the NuPage buffer with Coomassie G250 in it? It says that will create a dyefront that runs closer to the ion front than bromophenol blue...thus some smaller proteins/peptides which are cut off with bromophenol blue will not be cut off with G250.

But I can't find out what size of peptide this would make a difference for. Does anyone know exactly where, in terms of "kDa", these two dyefronts normally run?

(also, I assume G250 is some sort of special formulation of Coomassie that doesn't stain proteins...)

Thanks!

problem in the GST fusion protein purification

Thu, 05 Nov 2009 23:33:35 -0800

hi all,

Recently I met a problem in purification of GST fusion protein, the protein is expression and soluble, but can not bind the GST column, all in the flow through. I use the PBS(pH7.4) as the binding buffer, and elution buffer is GSSH(pH8.0). I already check purified ability of this column by run protein from pGEX-4T1 empty vector, and everything is ok.

And then I tried the his-tag construct, also got the soluble protein , but can not bind the HIS column.

The PI of my protein is 9.5, so if should I change the binding buffer's pH?

Everybody, Please recommend to me how to solve this problem.

Thank you very much

mervyn

PDI western blot

Thu, 05 Nov 2009 18:43:13 -0800

hi,

Please help. i have an antibody from santa cruz sc-17222 that should detect a band at 55kDa for PDI. PDI is present in liver so i am trying to use it as the positive control tissue, but i get the strongest band at ~110kDa, another weaker one at ~72kDa and the weakest one at ~55kDa. I always get three bands, no matter how long i denature or whether i use DTT or mercapto in the loading buffer. The band that i want is always the faintest one too.

I have seen in some papers that people use iodoacetamide to block the formation of dimers, but i have never tried this before as i have not come across this problem before?

Thanks

rachel

how to deal with protein precipitates during dialysis

Wed, 04 Nov 2009 17:18:09 -0800

Hi all:

I am now trying to purify my His-tagged protein. Some of the protein precipitated during dialysis, my next step is to cleave the protein using EKmax protease. It seemed like that the protease did not digest the precipitated proteins. What should I do with those precipitates? Any suggestion is appreciated!

iTRAQ cost compared to a DIGE approach

Tue, 03 Nov 2009 01:40:50 -0800

Dear all,

I�m thinking about doing a non-gel based technique approach to identify cell wall proteins (most of them are basic and highly glycosilated). Because of these characteristics, proteins doesn�t go easily inside of isoelectric focusing gels, so we will lose a lot of such proteins in a 2D approach.

I�m thinking about using iTRAQ technology, but until this point I don�t know how high would be the cost for analyzing 3 control and 3 treated samples. Do someone of you know, if the cost of such non-gel based techniques are much higher than a usual DIGE experiment? I refer to the cost of chemicals.

Thank you for your support.