Author Topic: Need help in troubleshooting  (Read 11568 times)

roy_arnab

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Need help in troubleshooting
« on: October 02, 2012, 08:19:27 PM »
My 2DE gels have long horizontal streaks concentrating at the acidic region. Previously, I had the same problem. But after following Sjouke's suggestion to avoid clean up kit (my sample is E. coli whole cell extract) and washing the cells in good purity ice-cold water I almost got rid of the problem and managed to produce good gels. Although, there was streaking noticeable on the acidic region (fig1: 7 cm pH4-7).

But, now, after we shifted to the new lab the streaking has come back. At first I was thinking about dust, but, i am taking every precaution to avoid dust. The surprising thing is every now and then 1 in 10 gels would come very nice (Fig2: 7 cm pH5-8), which makes troubleshooting more troublesome! Since I want to figure out a way by which I can produce consistent good quality 2DE gels and my 7 cm IPG strips are almost over, I am standardizing on 17 cm IPGs (pH4-7). On this IPG I am getting consistent streaking (Fig3: 17 cm pH4-7).

Sample preparation:
1. Cells grown to OD(595)=0.6, 20 mL culture spinned, washed with 20 mL ice-cold water four times.
2. Cells transferred to micro-centrifuge tubes.
3. To half of the pellet 500 micro L Rabiloud buffer was added (7 M U, 2 M T, 4% CHAPS, 10 mg/mL DTT & 0.5% Pharmalyte)
4. Flash frozen in liquid N2, allowed to thaw on ice.
5. Sonicated to break DNA strands (19 sec pulse, 50% duty cycle, 1 minute cooling, 15 times, always on ice-water).
6. Spinned hard 14,000 rpm for 20 minutes & supernatant stored @ -20 C.
7. After protein estimation (I get a concentration ~ 1-1.5 micrograms/micro-L) 120 micro-grams were loaded on IPG strip.
8. Rehydrated in passive mode (20 C) for 12 hours.
9. Strip was soaked on two damp tissue papers to get the oil out.
10. ~5 micro-L double distilled Milli-Q water was added to each of the paper wicks.
11. IEF was started.

I have tested two different IEF programs. In one case there was only one gradient (0 to 10,000 Volts, rapid, till it reaches the final V-hrs) and another with 3 steps (first: 0 to 250 volts, linear in 20 minutes, second: 250 to 10,000 volts, linear in 2:30 hrs and 3rd: hols till final V-hrs). I have tested two different final volt hours too (40 and 65 kV-hrs). But the results were not very different.

I do not throw the used oil. I store it in a separate bottle and use it again. Is this oil has some thing to do with streaking?
Please suggest what should I do!
« Last Edit: October 03, 2012, 12:22:45 AM by roy_arnab »

roy_arnab

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Re: Need help in troubleshooting
« Reply #1 on: October 03, 2012, 09:55:48 PM »
I have increased the concentration of Pharmalyte to 2% this time and limited the final Volt-hours to 50,000. Still to see the results!

I am looking forward to your suggestions please!

SlavkoM

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Re: Need help in troubleshooting
« Reply #2 on: October 04, 2012, 03:41:09 AM »
Hi,

Such streaking on 4-7 is very strange to me, I have never seen anything like it. I don't know about sample prep of bacteria, but is seems OK.

9. Strip was soaked on two damp tissue papers to get the oil out.

??? I don't get this. After rehydration, you should rinse the strip with dH2O to remove urea crystals if they formed (although I never saw any). Since you move the strip from oil (rehydration) to oil (IEF), I really don't see any point in removing the oil.

10. ~5 micro-L double distilled Milli-Q water was added to each of the paper wicks.

Only 5 microliters, is this a mistake? We throw paper wicks in dH2O so they are soaked completely, then put them on paper just to remove excess water (so it doesn't drip). We use IPGphor btw, I don't know if other IEF machines really use so little water on paper wicks. After all, paper wick has to conduct the current and collect charged impurities and proteins with pI out of gradient range, and for that it needs water.

Regarding protocol, we use standard recommended protocols with adding another low voltage step at the beggining. Something like this (18 or 24 cm strips):
300 V - 300 Vh (1h)
500 V - 500 Vh (1h)
gradient to 1000 V - 800 Vh (1h)
gradient to 10000 V - 16500 Vh (3h)
10000 V - untill end Vh (50-60 kVh for 4-7)

In my opinion, your protocol is too fast. Especially with rehydration loading, there is a high current at the beggining, so you should have a low voltage step to remove all salts and other charged impurities (they stack in paper wicks). Only then current drops and you can increase the voltage.

If the current doesn't drop below 10 microampers/strip after 500 V step, we prolong first 2 phases. I don't like it if the current is maxed during gradient to 10000 V (when most focusing takes place). This protocol has never ever failed to result in nice gel, and especially not in 4-7.

Regards and good luck,
Slavko
« Last Edit: October 04, 2012, 03:47:09 AM by SlavkoM »

roy_arnab

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Re: Need help in troubleshooting
« Reply #3 on: October 05, 2012, 04:02:52 AM »
Thank you very much, Slavkom! Yes, I have also searched for this kind of problem and learned that its not a common problem. Perhaps I am doing something very wrong.

The "ReadyStrip IPG Strip Instruction Manual" provided by Bio-Rad with the IPG strips mentions (Section 3.1, Point No. 10, Page-6) to remove the  unabsorbed protein by blotting on wet filter paper. Now, I understand that I was using Kimwipe tissue paper rather than a filter paper, can it be the problem? They have also mentioned that one can just drip the oil off by holding the strip vertically on a piece of filter paper.

I can try rinsing the IPG strip as you mentioned. But should I blot the excess water on a piece of filter paper after rinsing? Otherwise the excess water can dilute the concentrations of buffer inside the IPG gel!

If I don't remove the oil (since I am transferring from oil to oil) at least from the ends of the IPG strip, wouldn't it affect the contact of the wet paper wicks with the gel surface?

In Bio-Rad manual they have instructed to add 8 to 10 micro-L of nano pure/18.2 MOhm-cm water to each paper wick. But somewhere on the web I read that the paper wicks should not be wet, they should be damp. Anyway, I shall use 10 micro-L Milli-Q water next time.

Again, I have read that short IEF programs work best. Even the latest "ReadyStrip IPG Strip Instruction Manual" by Bio-Rad recommends to use only one step rapid gradient from 0 to 10,000 Volts for 40-60 kV-hrs at 20 C. In the previous manual they suggested the three step program that I mentioned in my post above.

I was especially bothered about "water-movement" caused by prolonged IEF runs and since I have seen my IPG strip looks dried up only on the acidic side after IEF run (I forgot to mention in my post), I thought I am unnecessarily prolonging my IEF program. Anyway, I can try the program you mentioned.

By the way, I just increased the concentration of Pharmalyte to 2% and I saw at least lot of spots (Fig4: 17 cm, pH4-7). But, still the streaking around the acidic region can easily be noticed. If we look closely, it seems that the Ef-Tu (the big large spot at the middle) is causing most problems, a thick line at the level of that spot is seen on the right side, as if some of the Ef-Tu is spread there. Do you think that I should decrease my protein load?

Looking forward to your valuable suggestions!
« Last Edit: October 05, 2012, 04:09:10 AM by roy_arnab »

SlavkoM

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Re: Need help in troubleshooting
« Reply #4 on: October 09, 2012, 07:42:16 AM »
Hi,

I was suspecting that you work on BioRad system because of all the differences in protocols. I am not familiar with those, so I can't say much.
Kimwipes instead of filter paper should not make any problems.
We rinse the strip then blot excess water on paper, just because it's in GE protocol which I follow since my beggining in 2DE. Oil at the end of strips shouldn't make problems with contact since you put wet paper on later (simplified to cooking leve :Dl, water goes together with water and oil goes together with oil). At least on our system, we sometimes leave oil in focusing tray and put new strips inside so they're completely covered with oil, then apply paper wicks and it works fine. Obviously, paper wicks on BioRad are much thiner because we put 150 microliters (by protocol) if we pipet solutions (in cases we don't use water) onto the wicks.
Regarding protocol, yes, it should be as short as possible, but as I said, I follow GE protocols. All of them have low voltage initial phases as I wrote above, the only thing that varies is the last step depending on required total Vhs. So, when they say the protocol should be as short as possible, it means you should not focus for 70 kVh if 40 kVh is enough. But it doesn't mean that you should ramp to 10000 V in one hour or something like that. But again, it seems that BioRad protocols are different. Water movement in prolonged IEF is a problem in very long runs (like over 100 kVh) so you shouldn't worry about that very much.
If 2 % Pharmalytes works and you don't have problems with high current (btw do you have your protocols fixed by Vh or h?), it's OK. In general, because Pharmalytes are charged, they contribute to current and that's why 0.5 % is recommended for 10000 V IEF.
On the other hand, if you see strip drying in acidic region, that is probably the cause of your streaking. But unfortunately, I have no idea how to fix that, especially in BioRad system which I'm not familiar with, but I doubt it's because of a too long IEF protocol.

Regards,
Slavko

roy_arnab

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Re: Need help in troubleshooting
« Reply #5 on: October 09, 2012, 09:04:53 PM »
Slavkom, Thanks again for your suggestions. Yes, Bio-Rad protocols and systems are quite different from the GE ones.
I am currently rinsing the oil and then blotting with a wet filter paper now a days after you have pointed out.
Yes, these paper wicks are thin. Should I try using a pair of customized paper wick made from filter paper which will be a little thicker?
Q: What solution do you use to wet the paper wick instead of water?
Water movement because of prolonged focusing should not be a factor for me then, as you mentioned, since I never go beyond 70 kVhrs in total.
My protocol is fixed by Volt-hours and not by hours. I did not have problem with high current as such, although sometimes because of the current constrain (50 mA/gel) the voltage does not reach 10 kV.

I thought that the drying of strip in the acidic region is because of DNA/RNA contamination in the sample. Sjouke suggested that if I am sonicating my sample during cell lysis then DNA/RNA should be broken in to pieces and it should no longer bother the IEF focusing. I am sonicating for 15-20 cycles, 20 sec per cycles, 50% duty always on ice and 1 minute cooling in between cycles. So, I am a bit puzzled!

researcher123

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Re: Need help in troubleshooting
« Reply #6 on: October 10, 2012, 04:54:26 AM »
hi, even i face this streaking towards the acidic side, at times...recently i did a 4-7 on liver cell line HepG2 extract, and there was streaking as well...dont quite know the reason though...am attaching a pic taken from the decyder analysis snapshot...


SlavkoM

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Re: Need help in troubleshooting
« Reply #7 on: October 11, 2012, 01:44:21 AM »
OK, I'm pretty much out of ideas what more you can try. Regarding paper wicks, I think those supplied by the manufacturer should work fine. Otherwise, all BioRad users would complain about them.
Just the answer about the solution: We use Destreak rehydration solution instead of water on cathodic paper wicks if we have a basic pH gradient (3-11 or 7-11) and overnight runs. It really improves separation in basic region. However, this has nothing to do with your problem and 4-7 gradient.

Good luck, Slavko

rachelo

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Re: Need help in troubleshooting
« Reply #8 on: October 12, 2012, 09:01:15 AM »
Hi,

     I mostly run IEF on a Multiphor, but I have a little experience with BioRad and IPGPhor systems.

     The BioRad paper wicks are VERY thin, with little capacity.  Some people change them every couple of hours early in the run to prevent them from drying out.  If the gel dries in a region it will streak.

     I almost never see urea crystals after IEF gel rehydration, so I almost never rinse my strips prior to loading them for focusing.  After a rinse, if the surface water is not completely blotted away, this, too could lead to streaking.

     Although the BioRad unit can apply 10 kV, I'm not sure that the gels look as good as when a maximum of 8 kV was employed.  I think many BioRad users don't use more than 8kV.

                   Good Luck,
                   Rachel

     

roy_arnab

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Re: Need help in troubleshooting
« Reply #9 on: October 19, 2012, 02:01:07 PM »
I appreciate your response Rachelo as much as I do Slavkom's too. I thought the streaking might be because of high amount of DNA contamination which might not be broken by sonication. I applied DNase/RNase before adding Urea/thiourea/DTT/CHAPS/pharmalyte. I am yet to see the results, but IEF did not run well. The voltage did not rich 4000 (7 cm, mini gel) and the streaking was as it was earlier. I am pretty sure that it is not going to come well. So, my assumption might be wrong. It was not DNA then.

If the paper wicks are causing the drying, I wonder why is it only at the positive end? Should I try making some paper wicks myself from whatman filter paper? Or should I try changing the wicks in between?

I have another doubt. Do you people use exactly the amount specified (125 micro-L for 7 cm & 300 micro-L for 17 cm)? Or you use a little more like 20-30 micro-L? Please let me know, I am really desperate to get rid of these streaking...

rachelo

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Re: Need help in troubleshooting
« Reply #10 on: October 21, 2012, 04:26:35 PM »
Hi again,
     Wicks & gels dry out on one side because of the direction of water migration due to electroendosmosis.  It should be OK to make your own wicks out of filter paper.

     I didn't notice earlier that you were using 7-cm IPGs; I don't have experience with them, but I thought GE used to specify a lower maximum voltage for focusing them, maybe 4 kV? 

     The volumes I use for rehydration are typically the ones that GE specifies for their strips.  I don't think that increasing the rehydration volume will cure the drying out problem.

        good luck!
        rachel

Sjouke Hoving

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Re: Need help in troubleshooting
« Reply #11 on: October 22, 2012, 12:40:31 AM »
Hi,

A lot has been said already. Most of the problems you describe should not occur on IPG 4-7. This is the most robust and easiest to handle gradient.
Did you have a look at the general protocols provided in the sticky topic. Even on high voltage instruments, try once the gradient has we use on the MultiPhor.
Another issue might be the quality of the thiourea... have you tried another batch of thiourea, this might cause streaking as well.

About the strip handling: after rehydration, just drop of the oil, don't play around with tissues or filter paper. The risk of damaging the surface is too big. You can even do the rehydration without the oil.
Place some thick filterpaper at the end of the strips, dipped in water and remove excess water by placing on some tissue (we used actually tap water).

Sjouke


roy_arnab

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Re: Need help in troubleshooting
« Reply #12 on: October 22, 2012, 06:42:34 AM »
Thanks again to both of you. I shall try your suggestions and get back soon! Thanks a lot!

roy_arnab

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Re: Need help in troubleshooting
« Reply #13 on: October 26, 2012, 08:05:09 AM »
Hello, I have attached the image of my latest 2DE gel. The changes I did are as follows:

1. Used a higher grade Thiourea (Sigma) instead of what I was using
2. Used the IEF program mentioned in the sticky note (3 hrs 300 V, 5 hrs linear gradient from 300-3500 V, 18 hrs at 3500 V)
3. Did not touched the gel after rehydration, just dripped off the oil by holding it vertically on a tissue paper.
4. Used paper wicks made from filter paper (Whatman filter paper No. 1) and soaked the paper with enough water (Milli-Q), excess water was removed by soaking in a separate filter paper.

The voltage reached 3500 V on time. The program also ran properly. But, the gel show the same streaking.

I ran another gel with DNase treated sample which also showed streaking (second image). Therefore, contamination of DNA is also not the cause.

Do you think the pH of my sample/buffer has anything to do with this? Should I adjust the pH by Tris-HCl?
By the way I have increased the concentration of urea from 7 M to 8 M.

Any suggestions please?

Sjouke Hoving

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Re: Need help in troubleshooting
« Reply #14 on: October 29, 2012, 12:40:41 AM »
Hi,
Unfortunately, there is not really an improvement visible. Standard solution for protein solubilization is 7M urea, 2M thiourea, 4% Chaps, 1% DTT and 1-2% Pharmalytes 3-10.
Can you go once more in detail through your sample preparation to see if there is an issue?

sjouke