http://www.protocol-online.org/forums/index.php Fri, 20 Nov 2009 17:59:10 -0800 360 http://www.protocol-online.org/forums/index.php?showtopic=11692 Hi.

I want to concentrate my cell lysates for western blot by precipitation of the proteins with trichloroacetic acid + acetone. I have some questions.

1. This treatment will not modify my proteins for the posterior immunoblot detection?

2. After the precipitation, what is the best method to store the lysates? Can I freeze the pellet? Or should I ressuspend it in some buffer or H2O and store it like that?

3. Can I quantify the protein concentration by the Bradford assay (as I usually do)?


Thanks in advance.
]]> Fri, 20 Nov 2009 04:24:06 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11692 http://www.protocol-online.org/forums/index.php?showtopic=11688 Hi

I would like to know if “non reducing SDS protein ladder” is available???

]]> Thu, 19 Nov 2009 22:48:56 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11688 http://www.protocol-online.org/forums/index.php?showtopic=11648 Has anyone used protein free blocking buffers before? I know Pierce sells one that is suppose to eliminate high background. My blots have high uniform background after I tried blocking in a non-animal protein blocker from Gilead Biosciences. Wed, 18 Nov 2009 09:04:55 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11648 http://www.protocol-online.org/forums/index.php?showtopic=11487 If I wanted to detect a protein that normally would be 9kDa as a monomer, and I got it biotinylated, would it be detected in a WB at a different molecular weight using Streptavidin-HRP for detection? And how much more heavier would it be?]]> Thu, 12 Nov 2009 07:44:49 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11487 http://www.protocol-online.org/forums/index.php?showtopic=11481
I expressed a fusion protein with mbp tag (MW: 81KDa+42KDa) and everything looks fine. Molecular weight from SDS-PAGE matches well with above. After the cleavage, it shows clearly a single band at 81 KDa and mbp single band (42KDa). After size exclusion column, I got the pure target protein (81KDa). However, from SDS-PAGE gel (16% + 5%), I got several discontinuous bands around 81KDa (they are really very close to each other). I know my protein is pure. I don't know the reason.
I made fresh APS solution and didn't make any difference. I tried to load different amount of proteins and the single bands occurs for smallest amount of samples.

The same situation happens for my another protein which has the molecular weight of 54KDa. But I haven't had the same problem before. Is there anyone who can give me some suggestions.


Thanks.

Haiying]]> Wed, 11 Nov 2009 21:34:28 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11481 http://www.protocol-online.org/forums/index.php?showtopic=11423
I am targeting HSP70 and since I was having some issues with the blotting, I decided to Coomasie Brillian stain the gel after the blot to check its efficiency. I loaded the gel like this:
1.Marker
2.HSP70 pure standard
3.sample 1(liver homogenate) total protein
4.sample 1(duplicate)
5.sample 2(liver homogenate) total protein
6. sample 2 (duplicate)
7. negative control (only sample buffer)
(etc as mirror until the 14th lane since the gel was to cut in 2 to test different antibodies)
I realized that my blot wasn't efficient since the proteins were still in the gel, but what I found strange was that the marker and positive control were in the gel, but from the liver homogenate ONLY the 70kD (which I assume is actually my target protein) were in the the gel. I haven't stained the membrane to see if something passed or not (although at least half of the marker passed). My question is, in a case of a bad blotting when the proteins stay in the SDS gel, a total protein homogenate should have many lanes and not only and exclusively on the 70kD. I use as extraction buffer 1 ml (of 1mMEDTA 1mM PMSF in PBS ice cold with protease inhibitor cocktail) for 100 mg liver (of fish by the way), homogeneized, centrifuged, and supernatant stored at -80C until sds page/western blot.

Any thoughts about this are very welcome since I think I am missing something very basic here...

My best regards to all

Anatg
]]> Mon, 09 Nov 2009 01:16:33 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11423 http://www.protocol-online.org/forums/index.php?showtopic=11422
I have a suspicion for why none of our bands look strong using polyclonal antibodies, even those for so-called abundant proteins.

We have a good scanner (Fuji LAS-3000) that we use for densitometry of EtBr-labeled DNA gels, which has emission filters at both 520 and 580. So we use it to visualize bands on Western blot by using fluorescently labeled secondaries (Alexa 488). Should we expect this to inherently lead to less sharp bands, or even to less intense bands, compared to the intensity of background staining? (this could be the tradeoff in which fluorescent-labeling being an easier and less messy procedure than HRP)

Any information you can give about experience with fluor-conjugated antibodies versus HRP- or AlkPhos- conjugated antibodies, for visualizing protein blots, would be interesting. Thanks.]]> Mon, 09 Nov 2009 00:25:34 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11422 http://www.protocol-online.org/forums/index.php?showtopic=11391 i was wondering if i can follow the same protocol for blotting as i do with PVDF.. except for the activation with methanol part of course...
but my transfer buffer also has 20% methanol.. can i continue using tat or remove it from my buffer for a NC??!!!
i usually run at 100V and 400mA max current for an hour!!!]]> Thu, 05 Nov 2009 20:24:31 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11391 http://www.protocol-online.org/forums/index.php?showtopic=11370
better if available in india.. loaly.. not imported.. cause i don have much time to wait for the shipment!!! ]]> Thu, 05 Nov 2009 01:26:50 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11370 http://www.protocol-online.org/forums/index.php?showtopic=11352 I'd really appreciate any help.]]> Wed, 04 Nov 2009 09:32:01 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11352 http://www.protocol-online.org/forums/index.php?showtopic=11344 i am using a molecular weight marker from Bangalore Genie (India)(Old catalog no. PMWM, new catalog no. 105979). In the image below A is the pattern which the company has given in the catalog, whereas B is the pattern which I got on same- 12% SDS-PAGE. As you can see clearly, there is a difference in the spacing between bands of the 2 patterns? Can anyone comment on why is it so? Is such case can I just count number of the bands and then compare the pattern order wise?
]]> Wed, 04 Nov 2009 04:19:32 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11344 http://www.protocol-online.org/forums/index.php?showtopic=11326 I've been using Amersham Full-Range Rainbow Molecular Weight marker as my ladder in my gels. However, this doesn't show up too well when stained with Coomassie Blue. Do you have any recommendations as to other types of ladders that will show up brightly? Thanks.]]> Tue, 03 Nov 2009 12:44:29 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11326 http://www.protocol-online.org/forums/index.php?showtopic=11308 So it was my first time doing a WB. Since I had to rush to an important thing, I did not have time to develop chromogen after 2ndry antibody. I was told that its OK to dry the membrane and later develop color, which I did. We then found out that nobody has ever done that and people dry either before they start the antibody washes or once its completely done, not in the middle. Is there anyway to salvage the situation? People told to try "activate" the membrane with methanol, wash it couple of times with PBS/TBS and then try developing color. But I wanted to ask the community if anybody has done that. By the way I'm using PVDF membrane.
Thanks in advance for your tips and assistance!]]> Mon, 02 Nov 2009 14:12:21 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11308 http://www.protocol-online.org/forums/index.php?showtopic=11298 I purified GST-fusion protein and checked by SDS-PAGE, bands were clear ,
but for western blot I think I have to dilute the GSTs , now I need to know the sensitive amount of it so that I can determine how many times the protein to be diluted.
thank you everybody, best regards to you!]]> Mon, 02 Nov 2009 02:25:35 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11298 http://www.protocol-online.org/forums/index.php?showtopic=11296
I´m new at this forum and have a little problem with a validation.
I used the search-function and couldn´t find any thread with this topic.

I have to validate the sds-page at work.
We are characterizing collagen typ I most of the time.
I never did a validation before so I read a lot and learned what to do.

So I get the information to vaildate a few important things.
Robustness is one of the easy to describe parameters.
I have big trouble in phrasing "Specificity" and a little trouble with "limit of detection".
About limit of detection: Made a series of measurements with descending percentage of collagen. So I can see at which level I can detect the last visible bands in the gel. Ok. But I have hassle to validate "Specificity". SDS-PAGE isn´t specific to collagen typ I, how to show that?
Any ideas?

My question: Has someone managed a validation for characterizing collagen typI with SDS-Page recently and is willing to help me with this?

Maybe this is a dumb question for some of you pros....

Thank you for reading and sorry for my english.

K.



]]> Mon, 02 Nov 2009 02:04:17 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11296 http://www.protocol-online.org/forums/index.php?showtopic=11284
I just began my bachelor thesis and have to extract a nuclear protein from mouse fibroblasts (to perform a Western Blot afterwards). Is it a good idea to use mouse ear tissue or do you have any better ideas? Maybe you have experience with that kind of experiment?!

Thanks in advance!
Isi]]> Sun, 01 Nov 2009 09:48:33 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11284 http://www.protocol-online.org/forums/index.php?showtopic=11270
I am not very good with calculations. I used the nanodrop instrument to determine my sample protein concentration as 2 mg/ml. I would like to add 20 ul into each well such that the final protein amount is 20 ng (per one well). Would any one please show in a step by step manner the calculation to derive this (I am confused with mg, ug, ng and the amount per well).

Since I need to load the protein in duplicate or triplicate, what would the calculations be for three wells: i.e 20 ul per well and there are three wells and the protein amount is still 20 ng per well. Remember, my original stock is 2 mg/ml. If this is too low, just use a suitable original concentration as an example. Please, can any one help me with this, as I am rather confused.

Thanks
lab2009



]]> Sat, 31 Oct 2009 08:15:23 -0700 http://www.protocol-online.org/forums/index.php?showtopic=11270 http://www.protocol-online.org/forums/index.php?showtopic=11254 Fri, 30 Oct 2009 04:47:50 -0700 http://www.protocol-online.org/forums/index.php?showtopic=11254 http://www.protocol-online.org/forums/index.php?showtopic=11236
I am doing a western blotting analysis under reducing condition while I suddenly found that the antibody is recommended to used under non-reducing condition. Is that will work or not? Has anyone tried that before? Thank you.

Xin
]]> Thu, 29 Oct 2009 11:14:48 -0700 http://www.protocol-online.org/forums/index.php?showtopic=11236 http://www.protocol-online.org/forums/index.php?showtopic=11231
I got a problem in separation of two high MW proteins after a co-IP. One of them is 220-230kDa the other one is ~190kDa. The problem arises because the smaller protein "diffuses" on 6% gel when I run the IP sample for a long time. This results in a black smear in the lane when I detect with the antibody against the larger protein on western. I guess there is so much of protein pulled-down that it is being recognized non-specifically by the antibody.
So how can I achieve a proper separation of the two peoteins, but avoid non-specific band recognition? Shoul run a 4% gel in the cold?
or use gradient gel? or swithch from Tris-glycine system to something else?

Well, please feel free to give any kind of advice - any input is greatly appreciated!

Happy Halloween by the way!

Dimilletronc
]]> Thu, 29 Oct 2009 09:31:34 -0700 http://www.protocol-online.org/forums/index.php?showtopic=11231