http://www.protocol-online.org/forums Tue, 07 Feb 2012 17:54:02 +0000 360 http://www.protocol-online.org/forums/topic/24320-birdlike-bandslanes-of-sds-page-and-wb/
I am really frustrated because I do not ge rid of these strange bands. Can you help me please?
For protein isolation I used the following kit: Total RNA and Protein Isolation from cell cultures - NucleoSpin® RNA/Protein (Marcherey-Nagel 740933.50 - starting material: cell cultures from human skeletal muscle cells). The protocol I followed as well as the pictures I got after PonceauS staining are attached. The same bands/lanes appear in the gel (Coomassie) - I did not add these pictures. The only difference I performed using this kit was that I used DTT in the lysis buffer instead of 2b-Me.
I run the samples in TrisBis Gels 4-12% (see pictures) and TrisGlycine 8-16% (both from Invitrogen) - but it remained the same. Furthermore I applied the samples in 25µL volume for all of them and used fresh running buffers.Is it too less or too much salts or too less TCEP - the reducing agent? I am at a loss...

Thanks a lot!

Stefanie
 help-WB.pdf (259.86K)
Number of downloads: 11]]> Tue, 07 Feb 2012 17:54:02 +0000 http://www.protocol-online.org/forums/topic/24320-birdlike-bandslanes-of-sds-page-and-wb/ http://www.protocol-online.org/forums/topic/24315-tris-hcl-becomes-cloudy/
I was preparing 1.5M Tris-Hcl pH 8.8, and the solution is cloudy, which I don't understand why. I tried to make it with distilled water & MilliQ H2O, the same. I tried to increase the temperature to 35deg C and dissolve, but still the same. pH was maintained at 8.8 throughout. I also put the solution under stirrer for a whole night and still the same. I didn't add any NaOH at all.
When I used this Tris-Hcl to run my buffer, the electrophoresis was fine. And even the Western Blot turned out pretty good.
But I don't understand the reason for the buffer to be still cloudy. Can anyone explain? Thanks.]]> Tue, 07 Feb 2012 11:06:50 +0000 http://www.protocol-online.org/forums/topic/24315-tris-hcl-becomes-cloudy/ http://www.protocol-online.org/forums/topic/24259-can-flag-ip-but-cannot-detect-with-western/
When I FLAG IP'ed my protein of interest, based on the silver stain compared to non-expressing cells, it seems to have worked (I intend to mass-spec). However, when I check the Western blot with FLAG antibody, I was unable to see my protein of interest (or rather, there's a nonspecific band located at the same molecular weight as my protein of interest that is found in both expressing and non-expressing cells).

There is an antibody detecting my protein of interest; it is able to detect it with other tags, just not FLAG. And this antibody does not pick up endogenous levels of my protein.

I have tried blotting just the cell lysates with FLAG antibody, but had no luck either. Prior to establishing my stable cell line, I verified that my cells still contain my transfected plasmid using RT-PCR.

tl'dr All this to say, I seem to have a working FLAG-tagged protein that can be IP'ed but cannot be detected with Western blot. Help?]]> Thu, 02 Feb 2012 18:34:30 +0000 http://www.protocol-online.org/forums/topic/24259-can-flag-ip-but-cannot-detect-with-western/ http://www.protocol-online.org/forums/topic/24231-my-protein-bands-and-protein-marker-becomes-abnormally-clearer-sometimes/
I´m having some problems with the resolution of my proteins in the gel. I have to check fosforilation in tagged proteins. It happens very often but not always. The day before I run some proteins nicely and when I do it again, with the same samples, it happens: protein marker is almost invisible in the gel, ponceau after transference is very clear, and I eventually obtain my protein very diffused and irregular when I develop the blot..., that´s why I ask u for help, to identify the possible variable/s. First I thought it was a problem of buffer, so I did some tests with different brands of different products ( glycine, SDS, Tris... Nothing changed. I changed even the water...again I didn´t observed any change,
My protein is about 150 kD of MW and I use a 10% ABA gel, made with a stacking buffer from Tris-HCl pH6.8 and a resolving buffer from TrisHCl pH 8.8. My running buffer pH is 8.6. I run 2 small gels at constant intensity of 30 mA. First the voltage is about 70v and later on, in the resolving part, it changes to 140v aprox.
I need to resolve this big protein and its phosphorilation bands, so I have to run the gel until the band that corresponds to 150kD is between 2/3 and1/2 of the resolving part, which means that I have to run the gel for 5 hours aprox., allowing the gel to run over 1-2 hours after the blue front run out. When I try less concentrated gels ( 8% ABA) never resolves the proteins nicer than 10%, and I observe the same problem indeed ( my samples and protein marker becomes abnormally clearer).
The transference is usually made in a transfer buffer similar to running buffer with 20% of methanol( at constant intensity of 400 mA along 40-60 minutes for 2 small gels).
Now the only thing I can do is to observe whether the marker ( I use 5 ul of thermoscientific pageruler) is quite visible in the gel or not...and then go on with my WB, but sometimes I have to do it 5 or 6 times until I get the proper resolution...and the problem is the degradation of my samples with the consecuent waste of time and money)
I measured all pH, even after running I measured the running buffer ( which did´t change from 8.6) cause I believed it was a pH problem. I read maybe it might be a problem with the resistance of the gel, which increases and consecuently would made increase the diameter of pore in the ABA gel and this could produce a protein faint.....

Any comment/suggestion/ criticism/ are welcome, thanks a lot for your help and your patience]]> Tue, 31 Jan 2012 16:16:41 +0000 http://www.protocol-online.org/forums/topic/24231-my-protein-bands-and-protein-marker-becomes-abnormally-clearer-sometimes/ http://www.protocol-online.org/forums/topic/24190-redoing-only-the-secondary-antibody-staining-on-a-western/
I was wondering what the thought was about redoing only the 2ndary antibody step on a Western Film.

During my first attempt at staining my gel, my film was too dark and I couldn't see individual bands very well.

The next time, I stripped the film, redid the primary step at the same concentration, and made my 2ndary 3X more dilute. When I developed the film, I didn't see anything. I'm guessing this was due to a low concentration of 2ndary antibody.

Since I didn't see anything from my film, I'm wondering if I could simply rinse off the membrane (to remove the developing solution, et. al) and simply incubate with a more secondary, this time at a higher concentration than my last round, but a lower concentration than my first.

Any input would be appreciated.]]> Sun, 29 Jan 2012 02:44:23 +0000 http://www.protocol-online.org/forums/topic/24190-redoing-only-the-secondary-antibody-staining-on-a-western/ http://www.protocol-online.org/forums/topic/24175-native-page-interaction-of-proteins-validity/
I am running a series of native PAGE experiments, using Bio-RAD TGX 4-15% gels, and Tris-Glycine buffer pH=8.1

My protein of interest has a pI of about 6, and I am investigating it's interactions with some multimeric proteins.

When running the experiment I usually prepare lanes as follows:
1)multimer alone
2)multime + protein
3)protein

I use silverstain for visualization.

I do not observe a direct colocalizaion of my protein on the multimers, but it surely changes its
pattern, and the protein in lane 2) does not wonder as far as the protein alone in 3).

What can I conclude from an experiment like this?

How du you like my setup for native PAGE in general?

And yeah, this is my first time with native PAGE.

best regards
Peter Jallerup]]> Fri, 27 Jan 2012 07:59:48 +0000 http://www.protocol-online.org/forums/topic/24175-native-page-interaction-of-proteins-validity/ http://www.protocol-online.org/forums/topic/24137-smeared-transfer-on-nitrocellulose-membrane/
I did two transfers in the same tank, while one membrane had perfect transfer, in the other the protein ladder was smeared across the membrane. Samples loaded on both the gels are the same (10% SDS-PAGE gels that I made). Before both the transfers I equilibrated the gels and membranes for around 5-10 minutes in transfer buffer. Transfer was done at 100 V for 90 minutes and left at 4C overnight. I prepared fresh transfer buffer with 20% methanol.
This has happened to me twice that one transfer is perfect while the second transfer is blotched . does anyone have a clue why this is happening?]]> Tue, 24 Jan 2012 17:02:54 +0000 http://www.protocol-online.org/forums/topic/24137-smeared-transfer-on-nitrocellulose-membrane/ http://www.protocol-online.org/forums/topic/24085-blocking-and-primary-antibody-in-different-buffers/
I decided to wash the filter in PBS-T 0.1% for while and reblock the filter in 5% milk instead og BSA.
I used the same aliqot of primary antibody diluted in 5% BSA overnight as previous western, and secondary in milk 5%.
DO you think its feasible and eventually could see my band in the film??
Or I should change something else??
Thank you]]> Thu, 19 Jan 2012 10:26:52 +0000 http://www.protocol-online.org/forums/topic/24085-blocking-and-primary-antibody-in-different-buffers/ http://www.protocol-online.org/forums/topic/24080-same-non-specific-band-for-different-target-proteins/ I have to quantify 3 different target proteins. but each time I do wetern blot, I get a very distinct and sharp nonspecific band above 100kD.
I have tried:
1. different chemicals form different vendors,
2. Primary antibodies obviously different
3. blocking both with BSA or milk
4. vectastatin secondary antibody (two anti rabbit, one anti mouse), avidin biotin complex-HRP conjugated (this is common for all runs)
5. developed with DAB (common for all runs)
6. even tried different tissues like brain tissue, retina, spinal cord
7. INTERSTING is that band appears same intense in all my 9 groups that is independent of total protein concentration (as ihave tried loading total protein with large differences).

What could be your opinion?
Thanks]]> Thu, 19 Jan 2012 07:10:17 +0000 http://www.protocol-online.org/forums/topic/24080-same-non-specific-band-for-different-target-proteins/ http://www.protocol-online.org/forums/topic/24077-types-of-96-well-plates-used-for-bca-protein-quantification/
How does the light (emission or absoption?) work in these machines?

Also, is a non-transparent one ok? or does it have to allow light to pass through the plate?]]> Thu, 19 Jan 2012 03:36:21 +0000 http://www.protocol-online.org/forums/topic/24077-types-of-96-well-plates-used-for-bca-protein-quantification/ http://www.protocol-online.org/forums/topic/24074-dot-blot-halo/
I have recently been trying to get a dot blot to work but instead of getting dots as the final result, I have been getting halos around where the dot should be. I placing 2 µL of sample onto dry nitrocellulose and using a 5% BSA in TBS-T. Also, the protein sample was obtained using a RIPA buffer (0.1 % SDS, 1% Triton x-100, 0.5% Na-deoxycholate). A manual spotting method is being used as we do not have access to a volume blotting unit.
I also did an experiment when I varied the amount of sample spotted from 1.5 µL to 10 µL.

If anyone has any ideas as to why I am getting this halo, I would be more than grateful.

Thank you]]> Thu, 19 Jan 2012 01:25:55 +0000 http://www.protocol-online.org/forums/topic/24074-dot-blot-halo/ http://www.protocol-online.org/forums/topic/24069-help-i-accidentally-use-transfer-buffer-as-running-buffer-what-will-happen/
I am so screwed up this morning. I am so worry right now.

I accidentally used transfer buffer as running buffer to run my gel this morning, and I found out and changed it back to running buffer in the middle, any of you has any idea what will happen? will it screw my gel????

My running buffer includes Glycine, Tris, and SDS, I took 80 ml and added 200 methanol, and 720 double distilled water

My transfer buffer includes Glycine and Tris only

Please let me know who do you think and if any of you has been through this before.

Hope to hear from you soon.]]> Wed, 18 Jan 2012 18:23:45 +0000 http://www.protocol-online.org/forums/topic/24069-help-i-accidentally-use-transfer-buffer-as-running-buffer-what-will-happen/ http://www.protocol-online.org/forums/topic/24060-western-blot-and-isoelectric-point/
Continuing with my previous problem. I found that all my proteins have pI [isoelectric point] more than 9. I read from milipore site that in western blot the pI can affect the protein transfer to membrane.

Methanol strips the SDS from proteins and proteins are finally transferred to membrane - I was wondering whether really the pI will affect the SDS-PAGE followed by western blot OR the pI is imp only if use the proteins in native form ?


Any one faced problems with proteins with high pI [Transfer buffer is pH8.3 and protein pI is more than 9 so proteins will have positive charge and will not migrate towards the membrane]]]> Wed, 18 Jan 2012 06:39:06 +0000 http://www.protocol-online.org/forums/topic/24060-western-blot-and-isoelectric-point/ http://www.protocol-online.org/forums/topic/24058-western-blot-developing-cassette/
Here in my lab are looking for some new film developing cassettes, but all the "official" ones are around 400 dollars each, in which we think we dont need them thaaaat much.

I am wondering if any of you had tried some more affordable brands with good success? Or could recommend any?

Thank you very much for your input!

Noe]]> Wed, 18 Jan 2012 02:26:47 +0000 http://www.protocol-online.org/forums/topic/24058-western-blot-developing-cassette/ http://www.protocol-online.org/forums/topic/24055-pull-down-assay-and-co-ip-experiment/ I have a problem with co-ip experiment. I identify the interaction between my protein and its target by pull down assay. Now i want to characterize this interaction by co-ip experiment but it isn't works. I use differents antibodies that recognize the proteins in different portions but i don't see the interaction. These antibodies works very good to precipitate my protein. I use different buffers but it isn't work. Can you help me? Do you think that the pull down works because the protein used as bait is to' mutch respect to' protein immunoprecipitate with the antibodies. There Are some solutions? Thank you]]> Tue, 17 Jan 2012 21:35:24 +0000 http://www.protocol-online.org/forums/topic/24055-pull-down-assay-and-co-ip-experiment/ http://www.protocol-online.org/forums/topic/24051-how-to-determine-whether-adenosine-a2b-receptor-is-inhibited-by-wb/
I need some help about how to determine if Adenosine A2B receptor is inhibited by its specific blocker MRS 1754 in H9c2 cells or adult-rat cardiomyocytes by using Western Blot? I cannot find specific downstream proteins to detect which are unique to A1/A3 receptors' which also locate in these types of cells.


Thanks!]]> Tue, 17 Jan 2012 17:12:11 +0000 http://www.protocol-online.org/forums/topic/24051-how-to-determine-whether-adenosine-a2b-receptor-is-inhibited-by-wb/ http://www.protocol-online.org/forums/topic/24041-trouble-detecting-histaq/

I want to do western blot for detecting 4 E. coli expressed proteins [size 23, 33,38,52 kDa]. previously i was not able to detect the proteins from induced cells. Then i purified the his taq proteins and was able to detect only the smallest band.

I was thinking the other proteins are high MW so did not transfer well - My transfer conditions are:

20% methanol - tris glycine buffer, 100V for 1.30 hrs - After transfer I could still see some high MW prestain marker bands on the gel. Primary antibody 1:100 for 3 hrs and secondary antibody for 1hr

I tried changing transfer buffer with reduced methanol to 12.5% and transfer time to 2hrs but the assembly got too hot - performed on ice - i could see vapours coming out - IS IT POSSIBLE THAT MY PROTEINS GOT DISTROYED BY HIGH TEMPERATURE

Also, I changed blocking time from 60 min to 30 min - fearing that my antigens could have been suppressed.


Right now I am thinking,
1. Could it be sample preparation - first time when i could see 23 mw protein the sample was 8 min heated but now i am using sample that is 15 min heated
2. The transfer assembly got to hot - and my proteins got distroyed
3. Changing methanol is not necessary for my proteins
4. Instead of using 2 hrs at 100V i can use overnight transfer
5. prolong the antibody incubation times]]> Tue, 17 Jan 2012 03:12:33 +0000 http://www.protocol-online.org/forums/topic/24041-trouble-detecting-histaq/ http://www.protocol-online.org/forums/topic/24019-western-gels-running-super-slow/
Basically I am running standard gels (10-12%), mini format, and they are taking many many hours (6-10+hours), sometimes not finishing ever. At first the buffer was getting very hot and I found out that I was putting the plates in the wrong orientation and didn't have a buffer dam (neither of these were issues with the precast system I used previously). Fixing these 2 issues fixed the heat problem, which was generating so much resistance that the current was maxing out at 400mA.

As part of troubleshooting, my PI suggested that a tech in the lab make a bunch of identical gels and we would troubleshoot in tandem to see what was wrong....when I ran one of his gels, with my samples, it ran perfectly...so troubleshooting over pretty quickly...

Now I made some gels and I'm back to many hours, but no excess heat/current. Of note, I was running 2 gels at the same time, one of which ran about halfway in 6 hours (and the ladder looked fine), and the other, the samples barely made it out of the stacking buffer in 6 hours.

Some of the things I've thought about:
- equipment...I took his gel box the most recent day I tried and still had a problem. I used the same powerbox as I used on the day my gel worked ok.
- the buffers...there is a lab common stock of running buffer, stacking buffer, resolving buffer...others are not having problems so its unlikely the buffer. The tank has plenty of running buffer, and isn't leaking out.
- my samples...don't think source of problem since they are just made by sonicating in PBS with inhibitors.
- number of gels at once - even though the boxes are designed for up to 4 gels at once, it have noticed that my successful run was only on 1 gel at a time. If running 2 simultaneously is a problem....??? any ideas,

Is there something I'm doing wrong in making the gels? That's the only other factor I haven't played around with. The acrylamide solution (we buy Protogel) is relatively recent...only a few months....the buffers are common. SDS is from lab common stock too. I used autoclaved ddH2O. TEMED is a few months old. APS isn't super fresh, maybe a month. My gels appear to have set properly in the normal amount of time...but could something else be going on?]]> Sat, 14 Jan 2012 23:30:53 +0000 http://www.protocol-online.org/forums/topic/24019-western-gels-running-super-slow/ http://www.protocol-online.org/forums/topic/24014-a-band-that-suppose-to-be-90kd-shows-up-at-70kd/ I have been coming a cross some unexplained blots. My target protein is suppose to be coming around 90 KD, but every time I run a WB it shows up at 70KD. I am following the general protocol for WB with very small changes. I called the company and they recommend to remove Tween from all of the buffers I am using, but this is didnt fix the problem and I am still seeing the bands at 70 KD.. any suggestion of why this is happening and how to fix it !!

MANY THANKS]]> Fri, 13 Jan 2012 20:50:59 +0000 http://www.protocol-online.org/forums/topic/24014-a-band-that-suppose-to-be-90kd-shows-up-at-70kd/ http://www.protocol-online.org/forums/topic/24007-extra-band-on-western-blot-of-insulin-recetor-beta-subunit-c-19-antibody/
Please help!]]> Thu, 12 Jan 2012 23:56:59 +0000 http://www.protocol-online.org/forums/topic/24007-extra-band-on-western-blot-of-insulin-recetor-beta-subunit-c-19-antibody/