I am trying 2-D gel electrophorosis. when I am doing that sample in normal 12% SDS page I am getting good band but in 2-D less spots than expected.... I am using GE helthcare 3-10pH strips. During IEF also I am getting good voltage running graph. i tried same sample 3 times and all time I am getting less spot. I am loading 1000 microgram of protein... But I am getting spots of 400 micrograms... so what to do? I checked every step Rehydration buffer Ampholyte every thing
Hi Yugantak, When you run the gel are the spots more dense in certain areas? The choice of gradient type or the range of the strip may be important.
Hi Yugantak
Some questions:
Did you observe white precipitated in the Strips after isoelectrofocusing?
Is the lysis solution composition the ideal for your sample?
Have you tried the cup loading method or the paper-bridge method for loading the sample?
1000 micrograms for a 3-10pH range strip is a lot of sample. I use to load about 100-150 micrograms for this kind of sample for silver staining
Is the sample clean of contaminants? salts, nucleic acids, etc....
Have you stained the strip after the 2º dimension to check if there has been a complete transfer of proteins from the strip to the laemmli gel?
Staining method you use?
B
Dear Gupta,
What is the length of the pH strips, 1000ug of protein can be loaded onto 24 cm strips (but not in 7 or 13 cm strips). Just make sure that you are not overloading the IPG strips.
Number of spots visualized depends on the staining procedure as well...
Did you observe any streaks or dense spot regions, particularly near the ends... if so you can try Narrow pH range (4-7 or 3-10 NL)...
What is the source of the protein sample... Try some protein sample clean up procedure prior to IEF.
Several factors could account for what you are observing. However, this problem requires a much more systematic approach to solve it. I would suggest you first confirm that you are actually loading 1mg by double-checking that the protein you are using to construct your standard curve during quantification of your samples is at the concentration you think it is. An incorrect standard could easily result in protein overestimation = meaning that what you think is 1mg is actually 400ug. If this is okay, I would next stain the IEF strip post-transfer to the second dimension. I have experience with certain samples (salt extracts of plant cell walls), which are not efficiently transferred into the second dimension using the normal equilibration buffer. For these samples I change the components of the equilibration solution in order to evacuate all the protein from the IEF strip. If you stain the IEF strip and see protein left there after running the second dimension, then that could account for your loss. Finally, check the sensitivity of your stain. Are your second dimension gels backed onto glass plates or not? Are you using Sypro ruby, coomassie, or silver? If you have an appropriate scanner I'd suggest using Sypro. If coomassie, then try colloidal coomassie instead of the normal coomassie.
If your sample contains a large number of different proteins and the sample has a wide range of abundances for those proteins, then it is possible that you are detecting only a small proportion of the proteins which are those in high abundance. There may be a large number of low abundance proteins which cannot be detected with your staining method but which together comprise a large proportion of the protein loaded.
@Patrick McHugh: No, Spot seperation was good.... only number of spot is less....
@Adrijana Leonardi: No mam.. Because after this I have to go for MALDI... So I am using only Coomassie colloidal stain
@Adrijana Leonardi: no precipitation, And I using standard composition which i read in so many papers. for loading the sample i simply pour the sample in Re-hydration tray... then over the sample strip (gel side towards the sample) the after 1 hour mineral oil...
I staining my gel with colloidal coomassie blue.
Yes before loading the sample i am doing 2D cleanup with GE Healthcare kit.
@Saravanan Kumar: I am using
18cm NL
no dence spot,
Yes before loading the sample i am doing 2D cleanup with GE Healthcare kit.
I staining my gel with colloidal coomassie blue.
@Stephen Chivasa:
I staining my gel with colloidal coomassie blue.
I stained my Strip after 2D its picking colour but how to check that protein is present in strip or not?...
Proteins in the IEF strip will appear as bands perpendicular to the long axis of the strip. If you just see a blue colour covering the whole background with no distinct bands, then it means the transfer from IEF to SDS gel was efficient. If this is the case, then please make sure that your initial protein quantitation is accurate. I am assuming that you are using the modified Bradford Method (in which you acidify the reaction), which is suitable for samples containing urea and thiourea, OR the Lowry Method. You need to be sure that you are loading 1mg protein and not less due to an overestimation arising from the protein assay. I suggest you repeat the assay with a fresh BSA standard.
@stephen Chivasa: I using normal Bradford,,, but sir last time i Did with liver sample.. in one shot liver gave very good spots.... in Kidney sample only I am facing this prob.....
In strip I am getting few vertical line at some places.. :(
It is not unusual that some protein fractions run nicely while others are problematic under the same conditions. As I said before, a normal Bradford is not linear if you are assaying proteins in urea/thiourea. In my Lab, we dissolve the proteins in 9M urea/2M thiourea/ 4% CHAPS. For Bradford assays in a final of 1 mL volume, we add 10 microlitres of 0.1M HCl to all the samples and the standards as well. The total amount of protein solubilisation solution (urea/thiourea/CHAPS) in every cuvette should be 10 microlitres in both the samples and standards. I use a 1mg/mL BSA stock solubilised in the same solution.
The bands you are seeing in the IEF strip is protein that has not transferred to the SDS gel. The more of this you have, the less spots you get in your 2D gel. Note that you do not always get 100% transfer. As you are dealing with soluble protein, in the first instance I would suggest you try mild ways to increase your transfer, such as to freeze-thaw the IEF strip once. In my group we routinely freeze the IEF strips at -20C overnight and thaw them just before equilibration. This enhances release of proteins.
@Stephen Chivasa: I'll follow your procedure.... I'll add HCL during the protein estimation.
Generally we are storing in -40C
@Stephen Chivasa: This time I add sonication also during homogenization.. and finally I stained gel in Silver stain to confirm that is protein is present in small concentration or not. but again I got negative result....... no spot.
- Badges
- Science topic