I ran a 2D-DIGE gel, with a pH 4-7 strip (NL, 24cm). The sample is transgenic mice serum, which has been removed from albumin and IgG(Albumin and IgG removal kits, GE), then ultrafiltered with 3kDa ultrafilter tubes (Millipore) and dissolved in lysis buffer( 7M Urea, 2M Thiourea, 4% Chaps, 30mM Tris-cl, pH 8.5) on ice and the protein concentration was determined by 2D-quant (GE). Then 25µg protein was dyed with 200pmol dye. Then 2X lysis buffer(8M Urea,4% Chaps,2% IPG buffer(pH4-7), 2% DTT) was added and the rehydration buffer was used to make the total volume up to 450µL. The conditions of IEF are: Stp 30V 12Hr, Stp 500V 1Hr, Stp 1000V 2 Hr, Grd 5000V 4 Hr, Grd 8000V 4 Hr, Stp 8000V 8Hr.Temp 20 ℃, 50µA/strip. After the IEF, the strips were equlibrated in 2% SDS, 30%Glycerol, 6M urea, 75mM Tris-cl 8.8, 0.002%Bromophenol blue with 1% DTT and subsequently 2.5 % IAA for 15 minutes, respectively. Then they were transfered to the 12.5% SDS-PAGE. The cathode and anode buffers were both the Tris-glycine-SDS (5X: 125mM Tris, 960mM Glycine,0.5% SDS, pH 8.3, what I used was 1X)buffer. 1W/Gel for 50min and 11W/ Gel for 5H40min. The current was 89mA and 225mA, respectively. The water circulation system set the temperature at 15℃. However, the Bromophenol blue was a line after the first 2 hours, and after the fourth hour of the SDS-PAGE , it became a curve and it was not very thin. And when the electrophoresis was over, the buffer is hotter than my hands. It seems the second dimension did not run well, but I attached the result, to see if anyone know what the problem is. Does anyone have any suggestions?
The problem seems to be the gel running. Have you used a running buffer without glycine? The presence of glycine ions is essential. Usually my running buffer is the following: TrisCl 250 mM/Glycine 1,92 M/SDS 1%.
I hope this can be useful. Good luck!
You may need to check your cooling system its seems to be not working since your buffer was very hot at the end of the run. it must be the increase of temperature during the run that induced the changes in your gel!
It seems that your gel pH is not adjusted properly so that your "stacking" is not working. The second dimension of 2D electrophoresis is based on the "Laemmli" system which is a discontinous gel system. In 2d the equilibrated IPG strip is acting as a Stacking gel and should have the same characteristics like a stacking gel in the laemmli system, namely a pH of 6.8! The resolving SDS gel should have a higher ph (i.e. 8.6) to increase the mobility of the trailing ion (glycine). Please change the pH of your equilibration buffers to 6.8, and check if the pH of your resolving Gel is between 8 and 9 (in our hands a pH of 8.6 works fine). With this adjustments your gels should look much better ;)
See
http://www.aesociety.org/areas/pdfs/Garfin_1DE_WebArticle9-07.pdf
(at 2.2 Discontinous buffer systems)
for a short description of the electrochemical phenomenon called stacking ;)
Please check the Focusing parameters also for ending with the precise spots. your gel is with inappreciable spots which could be manipulated with the focusing condition also.
Your 1st dimension focussing seems fine. I've ran 100s of 2D-gels and my equilibration buffer pH has always been of 8.8 just like your. Although Tris-HCl concentration in mine is 50mM not 75 mM. You do need a TGS running buffer (25mM Tris-HCl, 192mM Glycine, 0.1%SDS with pH of 8.3). Most important maybe is that you need to run your gels at a constant 24mA/gel (if running 2gels at a time then it's 48mA). You can maybe go as high as 30mA per gel but no higher. Gels will heat up otherwise and even cook on the plates.
I have never seen such a gel before, but it is probably due to a bad gel manufacturing or wrong concentration of components of Running buffer, you should check this parameters. First dimension and second dimension sounds good, so it is probably the gel or the running buffer. Good luck!
Your first dimension separation looks fine based on the high MW spots in the gel.
I second (third, fourth, fifth?) the opinion that you need to check your running buffer, and your running conditions for the second dimension. In addition to putting glycine in the buffer, you should also make sure to use higher concentration of SDS in the upper buffer if you are in a system with separate chambers.
I don't know what gel tank you are using, but for our DALTsix, the recommended temperature for the "cooling" water bath is 25 C for fast runs (5 to 6 hours), and 30 C for over night runs. Sounds counterintuitive at first, but makes sense if you think that you are trying to keep the temperature stable across the entire gel, i.e. avoid temperature gradients.
As an aside, it would be easier to assess you gel image if you showed us just one of the three channels (maybe the Cy2, if this is your internal standard, as in the typical DiGE setup). Make it a standard black-spots-on-white-background image, so that the contrast is better for the human eye.
I beleive the condition u r using for IEF running is not correct First of all make sure that u have prepared the sample well n u r geting the exact set point voltage bcoz if sample is having more salt concentration in that case u will not get exact set point voltage means no proper isoelectric focusing, n be very much sure that the gelside of strip is up n vic which r used in ends of strip are places corectly n d ammount of minral oil is sufficient. Once try conditions of IEF as: Stp 50V 2Hr, Stp 100V 1Hr, Stp 250V 1.5 Hr, Grd 7500V 2 Hr, Grd 10000V 4 Hr,
Hi Valeria, I am sorry for my mistake of the running buffer, actually it is Tris-glycine-SDS. I bought it from the company, it is 25mM Tris, 192mM Glycine, 0.1% SDS pH 8.3.
Hi Tammy-Lynn, thanks for your help. I am sorry for my mistake of the running buffer, in fact it is 25mM Tris, 192mM Glycine, 0.1% SDS pH 8.3.
Hi Elke, thanks for your help. I attach the cy2(standard) gel image here.
If your SDS-PAGE gel was casted by yourself, buffer concentration or pH of gel was not correct, too high or too low, I think.
I think your first dimension looks fine. The 2nd dimension is suspect. We have some times same type of gels. I agree with Tammy-Lynn when she says you should run your gels at constant current. In our DALT-Six system, we run 12-20% home made gels in a constant 21W current over night that have solves the problem of distortion of migration.
Hi collegues, I have found that the pump of the cooling system has not worked. The cooling water can not circulate. Do you think it is the most important problem that led to the failure of the gel?
I have used the conditions described above, it worked well. However, last time it failed.
I am eager to run a gel again. Someone told me that he did not use the cooling system while the result was still good. Should I follow his advice? Thanks in advance.
Greetings, Another thing to check is to make sure the upper upper buffer chamber in the DALT-6 isn't leaking. This importantly holds the 2X running buffer. If there is a slow leak, it can lead to spots looking like "tear-drops" due to the lower concentration of SDS. To solve it, make up molten 1% agarose in 2X running buffer and add a thin layer (after you've inserted the plates).. It only takes a couple of minutes to set. then add the 2X running buffer.
Possible reason is what u r saying failure of cooling pump; another; wrong conc. Of running buffer. As in high temp. The gel image tends to spread allover and doesn't come out fine.
hi Xiaojing
the temperature of running buffer is a critical point, so without a properly working cooling system the separation in the second dimension could be hampered. Other parameters seem to be ok. And now cross fingers waiting the new result!
Hi Xiaojing,
I'm agree with the majority that your first dimension seems okay. The problem is in the second dimension. If the concentration and the pH of the running buffer is fine, probably either the running condition or the gel itself.
For running condition, I also use constant ampere (24mA/gel for the separating gel). About the gel, Michitoshi Watanabe mentioned it before, probably you need to check the pH of the buffer (should be 8.8). And also, make sure that all of the reagents are mixed very well before you pour it into the caster and that the gel is completely polymerized before you use it.
I used to run my second dimension without controlling the temperature. People said that it is better to control it and make sure the temperature is the same, but we don't have the cooling system installed. And my gels seem to be fine. The running buffer feels a little bit warm after the running is over, but it's not really hot.
Good luck! :)
Hi Lisa, what you point out is really the key. The upper chamber buffer did leak. The solution in our lab is to make the height of the cathode and anode buffer in the same level. Since other guys in our lab always do it without anything wrong, I followed their protocol. This time I will try your solution. Thanks a lot.
Hi Collegues,
I have ran another 2D gel after the circulation system worked. This is the new result. It is really due to the temprature. I have been confused for one month and felt sorry for the waste of money. No matter what, now problem has been solved. It is lucky to get all your kind and professional help, which helped me go through it. Thanks a lot.
I have attached the new result here. And I will go on doing 2D-DIGE in the first week of New Year.
Sincerely,
Xiaojing Sui
Hi collegues,
I have performed 2D-DIGE again. This time I ran four gels with EttanDALT 6 unit (GE Healthcare). The tempreature of the system kept 15℃. The result seemed satisfying. Attached was the new picuture. The unsatisfying picture came from the six gels running together. I wonder if it was due to that when 6 gels ran together, the cooling system could not keep the tempreture at 15℃.
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