17 replies to this topic

[rachelhauser]

Hello everyone, I've been having the following problem:

I run 15% SDS-PAGE gels, at about 100 mA for 2 hours. When I do only 1D my samples show up fine, lots of protein bands. However, when I use IPG strips (3-10), do IEF overnight and run the gels, no spots appear... The colored MW markers run normally in the gel, I can see them fine, but no spots appear. I initially thought that maybe my agarose sealant was the problem, since it didn't solidify very well, but I made a fresh batch and now it's fine.

Common problems I have read about no spots in silver staining include:

- temperature (too low, or too high), which is not the case,
- wrong silver nitrate dilution (also not the case, and it's a new reagent from Sigma)...
- Using bicarbonate instead of carbonate for developing solution (also not the case...)
- the only problem I could think of was the thiosulphate, although I have used the same batch before and it worked FINE... and when I used NEW GE healthcare thiosulphate no spots appeared either...

What bugs me the most is that I KNOW the MW markers are there, and not getting stained!!!

Anyone help? Suggestion?

Thanks a lot!

Edited by rachelhauser, 13 August 2010 - 05:10 AM.

[yufang7]

You should post the detail steps of your experiment.
How much are you loading your samples?
How long were the IPG strips soaked in Equilibration buffer (including 1st and 2nd)?

[rachelhauser]

Hello, thank you for your reply... I loaded the limit according to the GE Healthcare Handbook, of 20 ug of protein per IPG strip. I also followed their 15 min rule for 1st and 15 min for 2nd equilibration, rinsed the strips a last time in 5 mL equilibration buffer rapidly and then sealed them with agarose and ran them at a constant rate.

I rehydrated them overnight, and did everything perfectly... is there any more information I can give you?

Thanks a lot... I'm hysterical trying to understand what may have gone wrong...

[yufang7]

What was the length of the IPG strip you used?
I think you can increase the amount of loading samples. GE Healthcare Handbook just is a recommendation. The amount of loading is based on the type of sample (from cells, tissue, membrane proteins, nuclear proteins and so on). If your sample is complexity (including thousands of different proteins) you should increase your loading amount. Furthermore, the length and pH rang of the IPG strip is the factor you should consider. pH 3-10 maybe need a high sample amount.
Samples from tissue 250 ug for 24cm pH 3-10 IPG strip (silver strain)
180 ug for 18cm pH 3-10 IPG strip (silver strain)

[rachelhauser]

Hello, are you sure about those amounts? pH 3-10 13 cm recommendation was 10-20 ug protein only!

Actually, I discovered my 2D quant-kit method leaves to be desired, and I think it is not very accurate, how do you quantify your proteins? Bradford, Lowry, kits?

I will try to up the amount of protein today, and let you know how it goes, but am still worried about my protein quantitation method...

[yufang7]

I quantify proteins using 2D Quant-Kit (GE Healthcare). Bradford has a bad compatibility with Urea, Thiourea and DTT. So I recommend 2D Quant-Kit for quantification of proteins dissolved in Urea buffer.

[rachelhauser]

actually, my proteins are biliary proteins diluted with milli-q water only... but u don't get much protein loss using that kit?

[yufang7]

There is not any problem to using the Kit for protein quantification.
How do you prepare your sample？
do the proteins precipitated and dissolved in lysis buffer (Urea buffer)? If not, it will make bad IEF results, because of salt, fat, saccharide… in samples.

[rachelhauser]

Hi, I use GE's sephadex columns G-25 with a exclusion size of 5 kDa to remove salts and other small interferents, diluting with water. And I don't solubilize them with and Urea, only with the solubilization buffer that GE recommends.. and no boiling either... But my GE 2D quant kit results give me extremely low protein values, and I think I may be getting some pretty big losses... but if u've never had a problema... I really don't know what else to do..!

[yufang7]

Hi

The following method may be helpful to you.


Bile fluid samples were sonicated and centrifuged at 16 000 g for 15 minutes at 4 ℃ to remove debris, nucleic acid and mucins as a preliminary separation. For sample delipidation, each 1 ml of preliminary separated sample was then mixed with 250 µl of CleanasciteTM HC (Ligo-Chem, Inc., Fairfield, NJ, USA) followed by rotation for 1 hour at 4 ℃. After incubation, each sample was centrifuged at 16 000 g for 1 minute to clear away the formed lipid-micelles, and the supernatant was transferred to a new tube. In order to reduce the salt concentration and other contaminants, a commercially available microcentrifuge filtration device (YM-3, molecular mass cut-off at 3 kD; Millipore, Bedford, MA, USA) was used to wash away contaminating species.

[rachelhauser]

Hi, I have that paper, did the same things, however, when comparing methods, found that desalting with G-25 sephadex columns yielded the best results...

If I don't know the exact protein concentration, would that be ok, I f I know it's order of magnitude, at least? In LC-MS/MS analysis, do I have to input the protein concentration exactly?

[rachelhauser]

Oh, another small question if I may... I have been trying out coomassie blue staining now, however, as the protein loads are much higher (100-240 ug of protein by GE recommendations), I have been having some problems with my focusing which I did not have when I used silver staining... I have run a 24 hour program, and my sample and dye have not reached the (+) end of my IPG strip... Is it ok to prolong the focusing at very high voltages for lots of hours? The program I use is:


step - 2 hs - 150 v
step - 2 hs - 300 v
step - 2 hs - 500 v
gradient - 2 hs - 1000 v
gradient - 4,5 hs - 10000 v
step - 4,5 hs - 10000 v
step - about 8-10 hours at 500 v, until I get to the lab and rmeove the strip for SDS-Page run.

Thank you so much again for your help.

[yufang7]

Hi

Will you process the sample by 1D SDS-PAGE LC-MS/MS? If you want identify different proteins as much as possible, you may load a more sample amount in 1D SDS-PAFGE. If you want identify the protein spots from 2DE, MALDI-TOF MS might be a better choice.

About your program, how much is the electric current at 10000V, when the step finished. And I suggest 8000 V or 6000 V for 13cm strip. dye have not reached the (+) end, it may indicate the sample is impurities (containing fat, polysaccharide et al. ). The Clean-Up Kit (GE) or TCA- Acetone precipitation may be helpful to sample purification.

[mdfenko]

rachelhauser, on 25 August 2010 - 03:44 AM, said:

Oh, another small question if I may... I have been trying out coomassie blue staining now, however, as the protein loads are much higher (100-240 ug of protein by GE recommendations), I have been having some problems with my focusing which I did not have when I used silver staining... I have run a 24 hour program, and my sample and dye have not reached the (+) end of my IPG strip... Is it ok to prolong the focusing at very high voltages for lots of hours? The program I use is:


step - 2 hs - 150 v
step - 2 hs - 300 v
step - 2 hs - 500 v
gradient - 2 hs - 1000 v
gradient - 4,5 hs - 10000 v
step - 4,5 hs - 10000 v
step - about 8-10 hours at 500 v, until I get to the lab and rmeove the strip for SDS-Page run.

Thank you so much again for your help.

you should not see the sample reach the end of the ipg strip (and why is there dye in the sample). the proteins will only migrate to the pH where their net charge is zero and should migrate no farther. the second dimension (sds-page) is where the dye should reach the end of the gel.

[rachelhauser]

Actually the dye is bromophenol blue, and is included in the rehydration solution in which I rehydrate my IPG strips... The dye should migrate to the + of the strip, even though proteins might reach their pI later... It is proff that a current is passing through the strip...!