- Mason Sweat added an answer:Why do I see my protein ladder standard on my developed western blot?
I'm attempting to use a western blot to detect the presence of an endogenous transcription factor. After I have shown that my western conditions are capable of detecting the transcription factor, I will use co-ip to show that the transcription factor is complexed with a separate protein.
Obviously, I need to be able to detect the protein in a western first!
1. Scrape cells from a 60 mm dish in 1 ml of pbs, pellet, aspirate pbs.
2. Lyse cells in Tropix lysis solution (ABX210LM), and add 1) general protease inhibitor (sigma) and PMSF (5 ul of each)
3. Add SDS loading buffer, boil 5 mins, load SDS-PAGE gell
4. Semi-dry transfer the gel onto the membrane (not nitrocellulose but the other kind, drawing a blank on that sorry). Ensure that my ladder standard is visible on the membrane.
Blocking: 10% carnation milk 1 hr room temp, slow rock
5. Primary antibody 4 degrees celcius ovn (1:1000, pbst)
3x wash pbst
6. Secondary antibody 4 degrees 1/2 hour (1:15000, pbst)
7. ECL prime, 5 min rt, exposed
8. blot dry and developed.
Any thoughts are appreciated!
Thank you Fabian, Tjerk and Raffaella. I am leaning towards using the 5% milk and the secondary antibody for 1 hr at rt. I will try the experiment again on Friday and update you all with my result!Following
- Sikder Nahidul Islam Rabbi added an answer:How can the resolution of bands be improved using agarose gel electrophoresis?
For the gel electrophoresis a 2 % agarose gel is made up (dimensions 20x20cm, 3-4mm thick). The gel is run for 4hrs at 85V. As running buffer 0.5x TBE is used.
A band pattern of 5-10 different bands (200-600bp) is expected depending on the sample. Some of the bands can be very similar in size and very close to each other. The gel image is not clear at all and the different bands cannot be distinguished.
Would high resolution agarose be a solution?
Image of your gel would be better to understand.i think ,you give more time to gel run,by that time your PCR product become out of the gel area.Gel chamber buffer volume is also a factor,better to give buffer 2 mili-miter over the gel level.From my daily experience with (HLA-Typing gel run),i have seen that,greater buffer volume over the gel, causing faint band or low resolution band,.
So,from my point of view,solution may includes
1.give less time at high voltage like 30 min at 150 Volt.
2.Adjust added buffer level during gel load.(not more than 2 or 3 mm) over the gel.
3.Check you Primer annealing temperature or Tm value of primer,for ensuring that you are getting right PCR product you wanted .Good Luck.Following
- Anyebe Onoja added an answer:What are the reasons a PCR that once functioned, is not working anymore?
Last week two Test-PCRs for a new set of primers worked quite well and i got one defined product band.
One week later i am not able to get any product again. I exchanged all materials, also the template (cDNA) i also tried reamplification from pcr product of the test PCRs.
Now i am wondering, why the pcr is not working anymore? Can Primers degrade within such a short time? Would it be senseful to run the primers on a gel electrophoresis?
Thanks for your suggestions and your answers!
Theresa, you do not have to trouble yourself. Definately, something is wrong along the line but you have to be systematic in finding out the problem. First, what temperature did you keep the primers? It will be fine at -20 to -80C, If kept at that temperature then the primer is ok. Next, is to ensure like Monica said to include a positive control and if the positive control does yield the expected product size, then you can suspect the reagents. That is if the positive control is properly kept at the right temperature (usually +4C before reconstitution and -80C after reconstitution). This way you will be able to tell if it is the template that has degraded. By the way, did you do a one step reaction or cDNA? Usually it is advisable to make cDNA as it is more stable, if you have to come back to the same template. Next, is to check your cycling conditions and be sure the conditions are same as was earlier used. Of primary interest is the annealing temperature, be sure it is exactly the same as before. Off course, you can run the template and the primer on a 2% agarose to see.Following
- Fuad Bahram added an answer:Can anyone help me in protein ubiquitination detection?
I'm currently working on a protein which is ubiquitinated for sure. Next step is to identify if it undergoes K-48 or/and K63 ubiquitination. I over-expressed my protein in combination with HA tagged Ub-K48 only or Ub-K63 only expressing plasmid, pull-down the target protein under denaturing conditions and then probe the sample with anti-HA antibody in immunoblotting. I always found a weak band corresponding to Ub(1) by using Ub-K48, and just one or two bands when Ub-K63 is present. I was wondering if anyone can help me to interpret these results. Dose anyone have an alternative protocol to assess ubiquitin moiety characterization?
Superfect should work fine much better than Ca-Phosph. The amount of plasmid also sounds reasonable...we used to transfect with 0.5-2.0 µg (depends on cell type, plasmid and size of cell culture). All parameters you mentioned are sensible but only thing I've standpoint, the amount of cell extract. You wrote that you were using six well plate.. Take a 5 cm dish / point.
- Nam Nguyen Nhat added an answer:Which buffer should I use for solubilization of hydrophobic proteins?I used a phosphate buffer but protein not dissolved. I attach 1D GEL scan. Bands are not resolved. There is lots of smearing.
I am new working in High density lipoprotein (HDL) which size 8-12nm, hydrophobic core structure with phospholipid layer outsite.
I want to disolve this protein to mono dispersation. Could anyone help me which solvent or buffer condition that help in this issue? Many thankful.Following
- Chinten James Lim added an answer:Alternatives to Trans-Blot Turbo PVDF packsMy lab has a Trans Blot Turbo from Bio-Rad and as far as I can tell you can only get the high speed transfers by ordering their proprietary blotting paper/buffer? I feel bad spending so much on the preassemble packages. Has anyone ever tried to get the rapid transfers on their own?
Excellent! Now I wonder if anyone has had luck recreating the Transblot Turbo buffer? We still use the provided buffer (with the Wypall X60) since each kit comes with enough buffer to last for 2-3x longer than after the provided stacks are long gone. Please post solutions here or redirect me to relevant post if applicable. Thnx!Following
- Marcel Imber added an answer:SDS-PAGE: Making freshly myself or buy ready-made?
Which is better - to make my own SDS-PAGE gels according to the available recipe, or buy the commercialised ready-made SDS-PAGE gels? Will the performance of the ready-made gels be lower than the freshly-made ones?
I was working in a lab where we always used ready-to-use gels and we never had a problem with that. It just depends on the money the lab has. If its possible it safes a lot of time and its more healthy for you because you dont have to work with TEMED and APS.Following
- Narayana Kilarkaje added an answer:What is the reason for problems with big errors after standardization of Western Blot results?
I am trying to check effect of degradation of some protein in cells after treatment with compounds. I performed few trials and I did standardization/normalization to loading control and to samples with my control compound - and trends every time are the same, but values differ a lot between each repeats... How is this possible? Some differences in f.ex. washing conditions or film exposure time should not affect WB after comparing to my controls, right?(but I try to keep always the same conditions)
When I try to calculate the average and calculate errors, th errors are extremely high even when results are in the same trend... Any idea?
One other thing. You can cross check your WB results by doing dot blot. If the latter is also giving no significant results, then the WB results are correct!Following
- Joseph Molnar added an answer:How can I be sure that the DNA ladder is separated well during gel electrophoresis?
I have made a gel electrophoresis for my DNA samples after PCR. The paper that I follow predicted that the size of the resulted two alleles is 292 and 247 and 447 for the outer primer as I used tetra primer arms method.
I have repeated the gel electrophoresis with different ranges of annealing temperatures (55-62) but the all the resulted bands were under the ladder ( i.e less than 100 bp).
I don't know if the ladder is separated well or not. I used agarose with 1.5-2%. I have attached the gel photos
Possibly your fragments run out of the agarose gel.
I guess that your samples are in linear forms but ccc or oc do not exist.Following
- Noha Hassuna added an answer:How do I name the different bands of coagulase gene ammplification?
Do I name them coa1 and so on or should I name them in another way as their sizes are different from those in literature
- Julijus Bogomolovas added an answer:Can I prepare a crude C. Elegans protein sample for protein assay (eg. by BCA or Bradford technique) and then use the same sample for SDS-PAGE?
1. How to prepare a protein sample for protein assay to determine the total protein content?
2. How to prepare the same sample for SDS-PAGE further?
3. Can I use Laemmli buffer to prepare the protein sample? (Will the buffer interfere the absorbance when measuring the total protein?)
We routinely use RC DC™ Protein Assay from Bio-Rad on our SDS-PAGE samples (mainly muscle lysates). This is a Lowry protein assay with preceding precipitation procedure. So you get rid of all interfering substances (reducing agents, detergents etc.) and then do the Lowry protein assay.Following
- Marcin Równicki added an answer:How can I detect 2'O Me RNA (13 bp) on gel electrophoresis?
I'd like to know, if anyone did this and how?
As a fluorescent tag, I tried to use Ethidium bromide and silver but it didn't work. I can't see any stripes on the gel. Any ideas?
Thank You Andrea and John for answers. The most important thing for me is how to detect 2'OMe RNA, because when I used EtBr it doesn't work. I will try with this SYBR gold.
- Fátima Milhano Santos added an answer:Which kit is more accurate for determination of protein concentration in SDS- Page sample buffer?
Which kit is more accurate for determination of protein concentration in SDS- Page sample buffer?
1- Protein assay kit (Bio-RAD)
2- 660 nm protein assay (Thermo scientific)
3- 2-D quant kit (GE healthcare life science)
Protein is extracted from lung biopsy and finally western blot will perform
2-D quant kit is the only one compatible with SDS because it applies precipitation before quantitation.Following
- Veronica De Pino added an answer:What is the role of PVP in CTAB buffer?What is the role of beta-mercuptoethanol?
- Niladri Kar added an answer:Any advice on western blot for ubiquitin?
I wish to perform western blotting for ubiquitin. In my earlier attempt I could not see the band corresponding to 8.5 kda in a 12% SDS gel. Please help me.Following
- Celia López-Herrera added an answer:What does "use in conjonction with an anti-mouse secondary against native antibodies" mean?
Thanks a lot to both of you!!
Mark, your answer has been really useful!
Thank you! :-)Following
- Kaid Johar added an answer:Does anyone use the Rictor antibody from Cell Signalling for western blot (Rictor (53A2) Rabbit mAb #2114)?
When I do the western, I get 2 bands between 171-238kDa, and I’m not sure which one is Rictor, because it should be around 200kDa. Anyone has experience with this antibody? If so, do you also get 2 bands? Thanks.Diana
We use same antibody but we get one band. You need to have longer time for transfer (about 2.5 hrs, 200 to 250 mA) and lots of cool packs around to not let temperature increase. We use marker that has a band at 250 kDa to confirm that transfer is complete.Following
- Katharine Wigginton added an answer:Does anybody know if there are any commercially available replaceable gels for capillary gel electrophoresis?I would require some commercially replaceable gels for capillary gel electrophoresis
Beckman Coulter offers a commercial replaceable gel kit for use in CGE. Here is some information about their product. The gel composition is proprietary, but the advantage of this kit is that it is commercially available. Several manufacturers of CE systems use this product and recommend it. Beckman also has their own lines of capillary systems.Following
- Jai Ghosh added an answer:Do I heat inactivate together with restriction digestion using NdeI and BamHI restriction enzyme sites?
I have a few questions about the use of NdeI and BamHI together in a double digest. For my insert, which has been generated via PCR, I plan on digesting it with Ndel and BamHI-HF (both from neb) for 2 hours, heat inactivate at 65C for 20mins, and then PCR purify.
For my vector backbone, I plan on digesting it with NdeI and BamHI-HF, for 2 hours heat inactivating at 65C for 20mins, adding TSAP (promega) and incubating at 37C for 1 hour, heat inactivation of TSAP at 74C for 15mins, and then PCR purifying.
The insert will then be ligated into my backbone, and transformed into chemically competent cells.
Although NdeI can be heat inactivated, BamHI-HF cannot. Therefore, is there any point in me heat inactivating the digestion after 2 hours? Also, after I have digested my insert and backbone, and TSAP incubated and inactivated the backbone, should I run these immediately on a gel and gel extract instead of PCR purifying?
Any help regarding this including suggested steps to follow instead would be greatly appreciated.
If you are aware of the reactions that these 2 enzymes catalyze then I would answer as NO. Do not heat inactivate both the enzymes together.Following
- Thu Betteridge added an answer:Why are there no free probe bands in my EMSA photos?
The EMSA photos attached to this question are taken by me. The free probe bands are so rare. It looks like the free probe were diluted by something. Almost a hundred EMSA photos have been taken by using the same kind of probes. No free probe bands are correct.
Which step of my procedure or part of my reagents or samples may make this kind of situation happen?
Thanks anyway for concerns.
On a second look of your gel, another scenario may be possible, would your probe only give out signal only after forming complex with protein? that is why there is no signal for free probe. In this case, is the unlabeled probe is your probe without the tag? it competes effectively there (no signal), whereas the mutant probe did not compete with your labeled probe (signal remains). How's that?Following
- Norbert Bittner added an answer:In BN-PAGE, why do we need to replace the heavy blue cathode buffer with light blue cathode buffer, after the dye running to one-third of the gel?
I cannot find any detailed explanation regarding this question, please help!
In blue native PAGE, almost all protocols mentioned to replace the heavy blue cathode buffer (i.e. in my case, 0.02% Coomassie blue) with light blue cathode buffer (0.002% Coomassie blue), after the dye runs til one-third of the gel.
If possible, please attach some materials for me to refer to. Many thanks in advance!
I sometimes replaced it with buffer without any Coomassie just to better see the protein bands. You can also try to just load your samples with deep blue cathode buffer and use cathode buffer without coomassie. It worked for my samples but i was just looking for samll proteins and no membrane proteins at all..Following
- Andrew P Herbert added an answer:How can I determine the monomeric/polymeric state of a protein?
I have a protein ~200 kDa that is running around ~800 kDa on native PAGE. I need to determine whether the protein is a monomer, dimer, etc; I think that the protein is a dimer (the ~800 kDA on native is not very reliable and may be aggregated with other proteins etc). I am having trouble determining how I could identify the polymeric state of this protein. The protein can only be obtained in relatively small amounts from mammalian cells.
As has been said, native PAGE is not a good method for determining molecular weight so you need to use another method.
Of the methods mentioned above:
DLS is probably not going to be accurate enough for you, and if you have very small amounts of larger aggregates/oligomers these disproportionately scatter.
SEC MALS could be a relatively simple method provided you can get enough material.
AUC would probably be ideal in this case. Check which type of optics you can use interference optics are good for proteins that don't absorb too well, bit if as you say the extinction coefficient is high then absorbance optics would probably be the perfect choice. I would suggest you try sedimentation equilibrium as this should easily give you your oligomeric state and also if there is an equilibrium between momoner and dimer for example you should be able to calculate a KD for this interaction.
Gel Filtration is shape dependent and therefore can often give misleading results for elongated proteins.
Hope this helpsFollowing
- Ofori Atta Linda added an answer:Does anyone have experience using soy milk as a blocking agent for IR western blotting?
We recently started using a Licor Odyssey Fc machine and got great results with the blocking buffer that Licor provides with the machine. However, we have used almost all of it and it is really expensive...so we tried our old recipes we used for our kodak imager/chemi that is simply non fat dry milk, tween, diH20 and TBS. Unlike previous results with the Licor BB (pure black background with distinct bands) my bankground was extremely red. I started researching other alternatives such as fish gelatin etc and came across soy milk...anyone have any experience with this???
Soy milk really work perfectly on immunoblottingFollowing
- Terence Spencer Crofts added an answer:Have you ever seen the pipette tips for cutting from the gel?
My advisor told me he had some sheet with ad for pipette tips used to cut bands from gel. However he didn't have it anymore so he couldn't tell me the official name nor brand.
Have you ever seen anything like that?
I just ordered some and will be trying them out in the not too distant future. In terms of length, they cover the length of our gel combs so that part is fine. The width does sound small, but it depends on if you want purity or yield more. Then smaller width means you are maybe getting the center 80% of your band and no neighboring bands which is what I am hoping for in my case.Following
- Sanjeev Bhardwaj added an answer:Among gel imaging systems, what advantages does the Bio Rad XRS+ have over the UVP biospectrum 510 system?
The XRS from Bio Rad is a little expensive for us but it has been widely used by other labs. What is the advantage of XRS?
The principle and working of both the systems are same.
However you have to decide whether the additional features like more sophisticated, less labor intensive, Multi dyes compatibility etc are worth for the additional cost or not!Following
- Nancy Duarte added an answer:Should Albumin be removed before 2DE gel electrophoresis from serum sample?
I am going to perform a 2DE gel electrophoresis from serum sample, before start a 2DE; will I remove albumin protein in serum sample? Is this important step? If anybody know help me.
Well, if you remove the most abundant proteins of the serum (albumin and immunoglobulin) you are going to improve the resolution of the less abundant proteins because in the 2D. Just imagine a huge amount of albumin in the gel, that giant spot is going to hide some other proteins of similar molecular weight and isoelectric point, so that if you remove albumin you will be able to discover many more spots in the same area that belongs to albumin.
There are many easy to use mini chromatography spin columns to remove in few steps the most abundant proteins (albumin and immunoglobulin), so if you have the chance to use one of those with your serum samples, go ahead and do itFollowing
- Lori Stevens added an answer:Is mixing 2 DNA Samples a problem?
I have 2 DNA samples with banding patterns (after doing PCR and electrophoresis) like this (number indicates the order of band):
Sample 1: 1 2 3 4 5 (miss 6)
Sample 2: 2 3 6 (miss 1 and 4)
As I mix these 2 DNA, I should have totally 6 bands. The result was that I just got 1 4 5 6 (miss 2 and 3). Can this situation happen (as 2 and 3 present in both samples).
PCR was carried out with RAPD primer. All the bands above are reproducible bands (because I have done PCR several times and just picked reproducible bands).
For the mix: I need 3µl DNA for 1 PCR reaction. Thus, I take 5µl DNA of each sample, mix them together, and take out 3µl from the mix to do PCR.
Thanks for your help!
RAPDs are hard to reproduce and the dynamics are changing with the DNA template.Following
- Masoud Maleki added an answer:What is your easiest loading buffer recipe?I need to make some loading buffer for my gels, and I have never made any before. We use bromophenol blue, and make a 6x buffer concentration. I am just doing simple 1% agar gels with ethidium bromide to look for DNA. If you have a simple recipe you could share, it would be greatly appreciated.
Elizabeth E Traylor
What sort of gel do you work with?Following
- Chunxi Zeng added an answer:What is the smallest detectable RNA fragment size in capillary electrophoresis?
I am trying to do structural probing on fluorophore labeled RNA by capillary electrophoresis. This is similar to DNA fragment analysis but no PCR is needed. The available equipment is Life Technologies 3130xl genetic analyzer with POP-6 polymer and a 50cm capillary array. My RNA is short (only 30 nt) so RNA fragments after in-line or enzymatic cleavage range from 1 to 30 nt.
I think some smallest RNA fragments will run out with buffer front so can't be resolved. But I am not sure about the threshold.
I am also wondering how much fluorophore labeled RNA should be loaded in one capillary. I am considering ROX or rhodamine 6G with decent quantum yield.
Your information can help me get started on this experiment. Thank you for your reply in advance.
Thank you, Ludovic!
It's interesting to know short capillary can decrease loss of short fragments. Do you know why? Retention of small fragments by polymer?Following
- Sandeep Ameta added an answer:What are the best conditions for getting separation between different conformations of HIV-1 RNA, using gel electrophoresis?
Currently I am trying different concentrations of HIV RNA, 1um, 4um, 6um, and 10um. I'm wondering if anyone has tried this perhaps in another RNA virus model and had success; by changing pH or Magnesium concentration?
In normal conditions you have usually formamide in the loading buffer which is also a denaturing agent...in non-denaturing loading buffers (you have glycerol instead (the one which is used for agarose gel...check 6X loading dye from ThermoScientific). Both contains same gel tracking dyes (Bromophenol blue and Cyanol).
So if you use non-denaturing buffer (commonly called as loading dye in many labs) you are sure that native conformation of RNA is not tampered by loading buffer..Following
About Gel Electrophoresis
Gel electrophoresis is a method for separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments, based on their size and charge.