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Sprag's Content

There have been 44 items by Sprag (Search limited from 12-November 18)



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#11961 Western blot - Stripping and Reprobing

Posted by Sprag on 08 March 2005 - 06:36 AM in Protein and Proteomics

If the different proteins are very different in size, and you are familiar with the antibodies (i.e. tried them individually on beforehand), I wouldn't bother stripping. Just use one at them time...
I use ReBlot from Chemicon and it works fine (the composition is obviously a secret...)

good luck



#10889 Need HeLa cell extract prepartion protocol

Posted by Sprag on 08 February 2005 - 09:56 AM in Protein and Proteomics

There are many different lysis buffers for mammalian cells dependent on which proteins you are interested in.
1% Triton X-100 in 50 mM Tris (pH 7.5), 150 mM NaCl + protease inhibitors is the most basic one.
Alternatively you can use 0.5% NP-40 in 50 mM Tris (pH 7.5), 150 mM NaCl, 10% Glycerol + protease inhibitors..

The method is widely used for identifing new binding partners, but it is VERY important to get your GST-fusion protein to very high purity, so you can be confident that what you pull out is truely binding partners and not some left overs from the purification. Always use GST alone as a control. Of course these interactions have to be verified by other methods before you can truely believe it.

have fun...



#10888 Protien expression

Posted by Sprag on 08 February 2005 - 09:47 AM in Protein and Proteomics

I don't think the concentration of IPTG, when using 1mM, matters for increasing the expression.
Instead, try to induce at higher density (OD600 > 0.6), or induce for longer time (overnight at 30 degrees). Or use a different strain. Some strains are optimized for expression of mammalian proteins.



#10887 GST fusion protein quantitation

Posted by Sprag on 08 February 2005 - 09:42 AM in Protein and Proteomics

The easiest way to quantify the amount of GST-fusion protein is to compare to BSA standards of known concentrations i.e. 3 microgram, 6 microgram and 10 microgram on a coomassie stained SDS-PAGE gel. Use equal amount of GST-fusion protein even though itís not completely pure.

good luck...



#10834 Bands on Western are not sharply defined

Posted by Sprag on 07 February 2005 - 04:46 AM in Protein and Proteomics

... and

You can get good bands in SDS-PAGE, if you cast the stacking gel fresh and make sure your buffers have the right pH....



#10833 Bands on Western are not sharply defined

Posted by Sprag on 07 February 2005 - 04:43 AM in Protein and Proteomics

That's the problem with SDS lysates, the SDS reacts with the DNA
Just sonicate your samples for 1-2 s... or add DNase to your sample buffer..

good luck..



#10017 Electrophoresis

Posted by Sprag on 14 January 2005 - 11:44 AM in Cell Biology

Maybe you are not adding enough protein on your gel?!.. Ponceau is not as sensitive as WB!!!

Also check if you really use nitrocellulose and not PVDF!!..

I've had problems with ponceau stainings of PVDF and instead of the standard Sigma (0.1% ponceau, 5% acetic acid), i made my own ponceau as 0.2% ponceau in 3% TCA, and that worked.



#10016 western blot

Posted by Sprag on 14 January 2005 - 11:38 AM in Protein and Proteomics

I would assume there's something wrong with your sample prep, as your transfer seems to work according to the markers; if the marker is there, the protein (if any) has been transfered.

try to measure the protein concentration in you sample(s) before you run your gel.

good luck

ps..There's nothing wrong with your transfer buffer...



#9521 phosphospecific antibody

Posted by Sprag on 21 December 2004 - 06:12 AM in Protein and Proteomics

It's not easy to make phosphospecific antibodies, and honestly, I would recommend getting a company to do it for you, and thus guarantee the quality and specificity. But otherwise, yes, you have to start off with a phosphopeptide, as using phosphorylated protein will most likely produce antibodies to other sites as well as the phospho-group.
Alternatively, you could test phosphotyrosine, -serine, and -threonine antibodies which are available. one of them might work with your protein.

good luck



#9295 affinity chromatography

Posted by Sprag on 09 December 2004 - 08:48 AM in Protein and Proteomics

Agarose and sepharose is the material the baeds are made of.
I think agarose is from Sigma, whereas sepharose is Amersham,... but pratically it's the same, though I personel prefer sepharose as they are easier to see...



#9236 EDTA only for cell dissociation

Posted by Sprag on 07 December 2004 - 08:51 AM in Cell Biology

Yes, you can use EDTA only, but it's usually not very efficient, that's why you have the combination.



#9235 Cell fixation

Posted by Sprag on 07 December 2004 - 08:49 AM in Cell Biology

well, that depends on the antibody. Some antibodies works best with methanol fixation, some with PFA. This is usually specified in the spec-sheet it's a commercial antibody, otherwise look up previous publications using that specific antibody, or try different combinations yourself. Another problem could be whether the epitope is intra- or extracellular. If intracellular, you also need to find the optimal permeabilising agent (methanol, TX100, saponin, etc).

good luck,



#9234 densitometry of westerns

Posted by Sprag on 07 December 2004 - 08:40 AM in Protein and Proteomics

you can use Adobe Photoshop (well, I know it's not free, but just mentioning it in case you have it already), as long as you keep the bands i.e. boxes with ALL yours sample, at similar sizes...



#8898 Storage of 30% bis/acrylamide and SDS-PAGE gels

Posted by Sprag on 25 November 2004 - 09:47 AM in Protein and Proteomics

for good resolution on your PAGE, you should cast the staking gel fresh. This way you get the best shift in pH from the staking to the resolving gel, and that's the critical factor for getting really good sharp bands.
You can easily cast your resolving gels and keep them in the fridge, as long as you keep the gels moist so they don't evaporate as suggested, and itís not a problem to keep them for a week or so.

good luck.



#8896 Primary antibody

Posted by Sprag on 25 November 2004 - 09:37 AM in Protein and Proteomics

well, the minimum time for primary can be as low as 20min, but i do recommend 60min. The washes, I do 3 times 20min in a 30-50ml for a minigel, but you can do shorter times, depends on your antibody. Nice western is a balance between high signal and low background, so if you get no background at all, you can easily shorten your incubation times, whereas higher background, you can either do longer washes, higher salt/tween/milk, or you can lower your concentration of primary...

have fun,



#8811 Odd Western Blotting problems

Posted by Sprag on 22 November 2004 - 09:12 AM in Protein and Proteomics

I'm not sure why you air-dry your membranes before incubating with primary. I normally block the membrane in 2.5%-5% milk for 1h before I incubate in primary.
Also, I wash 3 times 20min both after incubating with primary and secondary...

Try that....

good luck



#8760 unstable stables

Posted by Sprag on 19 November 2004 - 07:51 AM in Cell Biology

It's a very common phenomena that stable cell line looses expression especially if the gene is lethal. One trick is to express your protein of interest fused to a fluorescent protein such as GFP, so you regularly can FACS your cells and sustain high expression.

good luck,



#8758 How many cell passages to be safe ?

Posted by Sprag on 19 November 2004 - 07:39 AM in Cell Biology

some of the cell lines out there, such as HeLa, CHO, Cos-7 etc etc have been through countless number of passages. I would bring up fresh cells as soon as I see morphological changes, or if by mistake they've gone too confluent.



#8757 Cell fixation

Posted by Sprag on 19 November 2004 - 07:36 AM in Cell Biology

I'm not sure what the question is, but PFA works by crosslinking proteins, and thus fixes the cells. You can fix cells before FACS, as it's easier to handle and you can leave them in the fridge etc.



#8660 help - stable transfection clone pick up

Posted by Sprag on 16 November 2004 - 08:40 AM in Cell Biology

I don't use any trypsin,... just suck them up with the tip using your Gilson..



#8659 Precipitation with TCA and Na deoxycholate

Posted by Sprag on 16 November 2004 - 08:35 AM in Protein and Proteomics

deoxycholate binds to the protein and increases the interaction with the TCA. You usually add it for precipitation of very small amounts of protein with TCA..



#8658 Theory question on protein phosphorylation

Posted by Sprag on 16 November 2004 - 08:31 AM in Protein and Proteomics

I wouldn't think so, but the changes might be so minor that it can be hard to detect the differences... But no, phosphorylation should change both molecular weight (Western) and the pH (IEF)..



#8656 SDS-PAGE high MW protein trapped

Posted by Sprag on 16 November 2004 - 08:25 AM in Protein and Proteomics

maybe try to denature in 8M urea...



#8480 Western blot problem: not running in gel

Posted by Sprag on 06 November 2004 - 01:14 PM in Protein and Proteomics

If you are not to boil your sampes (?!) then try to dissolve in 8M urea... You need to completely denature your protein otherwise you'll see these dimers/trimers, and you can't really trust your markers etc etc...

good luck,



#8433 frameshift after ligation

Posted by Sprag on 04 November 2004 - 08:57 AM in Molecular Biology

So you've sequenced it, since you know you've got an extra base?!

You don't have to use CIAP when you are using two different enzymes... alternatively, you could put the pcr product into TOPO and then cut out from there. Then you'll know you've got the right ends...

Have you tried to cut with EcoRI only and thus seen a shift in weight?




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