http://www.protocol-online.org/forums Fri, 10 Feb 2012 14:04:50 +0000 360 http://www.protocol-online.org/forums/topic/24376-western-blot-loading-control/ When I am using loading control antibodies in my western blot (I am suing CDC-42 and GAPDH) I see multiple bands. CDC 42 has the MW of 21 but I see a very strong band at 43 kDA and more some not soo strong inespecific bands. GAPDH I also see inespecific bands. Can be because I am not using a protease inhibitor when I am extracting proteins. I am extracting proteins from Candida albicans. It is diffcult to fing a loading control. I found tubulin, the signal was perfect, no unespecific bands, but I am working with Heat Shock Proteins, so Tubulin is also activate tubulin, so I cannot use tubulin to control teh loading of my proteins. with beat actin I got a very weak signal and a very high background. Wating for an answer, please. Fri, 10 Feb 2012 14:04:50 +0000 http://www.protocol-online.org/forums/topic/24376-western-blot-loading-control/ http://www.protocol-online.org/forums/topic/24345-insulin-receptor-mobility/ What is the mobility of the insulin receptor in the cells? Does it connect to the cytoskeleton or is it freely mobile? Wed, 08 Feb 2012 22:38:11 +0000 http://www.protocol-online.org/forums/topic/24345-insulin-receptor-mobility/ http://www.protocol-online.org/forums/topic/24334-lysis-buffer-not-lysing-adherent-cells/
ok so I am new to western blotting (I know, I am a biochemist 2 years into a 4 year phd and never done a western blot before, half the rest of the lab cannot believe it either...) but I seem to have encountered a problem before I even start!

So I have my confluent cells in a 6 well plate, apply my conditions and timepoints, remove the media, apply 1ml ice cold PBS, remove, and apply 200ul lysis buffer. According to my fellow scientists showing me the technique, the cells should lyse quite fast and you should be able to see this under the microscope. However I look under the microscope and can still see my cells sitting there, fairly happily, still stuck to the bottom of the plate, still 10min after I have applied the lysis buffer.

In the end I used cell scrapers to scrape the cells off the bottom of the plate, and put the whole mixture, buffer, cell debris and all into eppendorfs and into the -80. But have no idea if this mixture is useless or what.


My main theory is that I am using adherent cells (HeLa, HMEC-1 (an endothelial cell line), and HUVEC (endothelial primary cells), whereas most of my labmates are pure immunologists and so are used to lysing non-adherent leucocytes.

Are adherent cells harder to lyse than non-adherent? or do they just look different when they do? or do I just need to use a different lysis buffer? people here doing monocytes or other leucocytes say that you can see the cells lysing under the microscope quite often and that it should happen fairly fast, are adherent cells just needing a slightly different technique?



Of course the first question any of you is going to ask me is what is the lysis buffer so here is the recipe I was given:

-50mM NaCl
-50mM Hepes
-1% Triton X
-1 tablet phosSTOP (phosphatase inhibitor)
-1 tablet protease inhibitors
-10ml H2O

(I am not sure of the brand of the tablets, this is my first time with westerns so just borrowing reagents from friend to get me off the ground for now)]]> Wed, 08 Feb 2012 11:38:41 +0000 http://www.protocol-online.org/forums/topic/24334-lysis-buffer-not-lysing-adherent-cells/ http://www.protocol-online.org/forums/topic/24305-properties-of-amino-acids-and-protein-functional-activity/
(Sorry in advance, if someone finds this question to trivial... I am not very familiar with this topic, but I have to get some quick answer.)]]> Mon, 06 Feb 2012 16:02:25 +0000 http://www.protocol-online.org/forums/topic/24305-properties-of-amino-acids-and-protein-functional-activity/ http://www.protocol-online.org/forums/topic/24294-co-ip-and-lc-mass/ I want to use lc-mass to analyzes my co-ip .
My first question is does the buffer of co-ip can be directly used in lc-mass. I hear the buffer of co-ip cannot be used with 2D gel.
My second question this how many compositions can be analyzed using the method(co-ip add lc-mass) and how much the loading quality needed.
My third question is whether two co-ip is needed[font=宋]？[/font][font="Times New Roman"] I see somebody use two different epitopes to ip the protein for example using antibody for FLAF tag and the protein own. Whether 2 co-ip need relatively more protein loading.[/font]
[font="Times New Roman"]What is lc-mass-mass? The advantage by adding additional mass. [/font]

thanks alot！]]> Sun, 05 Feb 2012 02:50:29 +0000 http://www.protocol-online.org/forums/topic/24294-co-ip-and-lc-mass/ http://www.protocol-online.org/forums/topic/24290-phosphorylation/ What is basal phosphorylation? Sat, 04 Feb 2012 17:18:42 +0000 http://www.protocol-online.org/forums/topic/24290-phosphorylation/ http://www.protocol-online.org/forums/topic/24285-protein-tagging-with-gfpeffect-of-expression-construct/
I hope this is the correct place to create the topic. I have a question about tagging a protein with GFP (fusion protein) to track its localization in the cells. Here it goes:

In almost every article researchers say, "we tagged protein x with GFP by using transiently transfected vector y" but they seldom explain the effect of expression levels of engineered protein and its biological effects.

If, let's say, I want to tag a protein whose intracellular level affects organelle morphology (for instance Mfn2 increases mitochondrial fusion) how can I achieve tagging the protein without flooding the biological system with excess amount of protein of interest (e.g. Mfn2). In other words, how can I tag protein of interest with GFP without overexpressing it?

transient transfection vectors are all containing strong promoters for high level expression of proteins of interest. This is good for studies involving protein isolation, but how can I tag a protein with GFP in the cells by transient transfection?

I hope it was clear enough to express my question.
Thanks for the answers in advance]]> Sat, 04 Feb 2012 06:44:58 +0000 http://www.protocol-online.org/forums/topic/24285-protein-tagging-with-gfpeffect-of-expression-construct/ http://www.protocol-online.org/forums/topic/24283-analyzing-western-bands-by-densiometry/ Thanks in advance -:]]> Sat, 04 Feb 2012 02:25:20 +0000 http://www.protocol-online.org/forums/topic/24283-analyzing-western-bands-by-densiometry/ http://www.protocol-online.org/forums/topic/24278-molecular-weights-of-insulin-a-chain-and-b-chain/ What are the molecular weights of human Insulin A chain and B chain?

Thanks,]]> Fri, 03 Feb 2012 18:12:38 +0000 http://www.protocol-online.org/forums/topic/24278-molecular-weights-of-insulin-a-chain-and-b-chain/ http://www.protocol-online.org/forums/topic/24277-suggestions-on-hard-to-express-proteins-in-e-coli/
since we are currently looking for proteins to challenge our protein expression platform i would be happy to hear your thoughts on proteins that are hard or nearly impossible to express in E. coli.

I would be happy to hear your thoughts on proteins that are hard to express (low yields, toxic genes) ...and where easy and fast downstream analytics (enzyme assay, ELISA, whatever) are available. I'm sure hundred of people here in this forum have wrecked their heads on expression of proteins ...your input would be highly appreciated.

Many thanks in advance!

Best regards,
p]]> Fri, 03 Feb 2012 17:13:18 +0000 http://www.protocol-online.org/forums/topic/24277-suggestions-on-hard-to-express-proteins-in-e-coli/ http://www.protocol-online.org/forums/topic/24273-y2h-screening/
I have a question about Y2H screening services. Has anyone used it? Which are the best companies? Is a tedious work? HOW MUCH does it cost? I found a lot of companies on the internet but would like to have a preliminary idea...

Thanks a lot.]]> Fri, 03 Feb 2012 13:23:00 +0000 http://www.protocol-online.org/forums/topic/24273-y2h-screening/ http://www.protocol-online.org/forums/topic/24271-zero-length-crosslinking/ Please share your experience.
Thanks,
Wishwas.]]> Fri, 03 Feb 2012 09:31:43 +0000 http://www.protocol-online.org/forums/topic/24271-zero-length-crosslinking/ http://www.protocol-online.org/forums/topic/24268-signaling-pathways-minutes-or-hours/
I have a theoretical question. I have seen many articles isolating protein lysates 15-30min after cells were treated with different growth factors (pathway activators) or common biochemical pathway inhibitors, such as LY294002 or PD98059. Some of them then link the alterations in the signalling pathways (e.g. PI3K or MAPK) to processes, such as cell migration, which sometimes take 20 hours to complete, but they do not investigate whether this pathway activation/deactivation is sustained throughout the whole 20h. Is this really a correct thing to do? Can you expect that a brief 15-30min activation of a signaling pathway can trigger behavioral or morphological effects on cells, which would last for 15 hours?]]> Fri, 03 Feb 2012 02:52:16 +0000 http://www.protocol-online.org/forums/topic/24268-signaling-pathways-minutes-or-hours/ http://www.protocol-online.org/forums/topic/24159-what-is-the-molecular-weight-of-fusion-protein-in-pet-32a-molecular-weight-of-the-fusion-protein-in-pet-32a/ I just start to use pET-32a expression vector and i need to know the MW of my protein after attached to trx Thu, 26 Jan 2012 03:52:48 +0000 http://www.protocol-online.org/forums/topic/24159-what-is-the-molecular-weight-of-fusion-protein-in-pet-32a-molecular-weight-of-the-fusion-protein-in-pet-32a/ http://www.protocol-online.org/forums/topic/24152-peptide-protein-conjugation/
Does anyone have a good working protocol for the conjugation of peptide to a carrier protein? (Doesn't matter if BSA, Ova, KHL). I need it for ELISA plate coating.
I think that the glutaraldehyde method would be best, as it is said to couple at the N-term of the peptide. But I am thankful for any suggestions.
I know the very best is peptide synthesis with an additional Cys at the N-term, but, well, peptide is already there - no Cys, so I will have to work with what I've got
Is there a possibility of influencing or at least checking the coupling ratio of protein:peptide?

Thank you all for help and support!]]> Wed, 25 Jan 2012 12:45:42 +0000 http://www.protocol-online.org/forums/topic/24152-peptide-protein-conjugation/ http://www.protocol-online.org/forums/topic/24129-meaningless-protein-protein-interaction-exists/ Tue, 24 Jan 2012 07:22:15 +0000 http://www.protocol-online.org/forums/topic/24129-meaningless-protein-protein-interaction-exists/ http://www.protocol-online.org/forums/topic/24082-kinase-assay-problem/
I'm trying to develop a non-radioactive kinase assay using MBP as a substrate. The plan is to perform the kinase reaction (using 50uM cold ATP - no hot ATP at all), then add sample buffer and run on SDS-PAGE, followed by western blot using anti-phospho-threonine antibodies. My problem is that I get bands that correspond to the MBP MW) even when no kinase is present. The signal does not increase with increasing concentrations of my kinase. I should say that the kinase is commercial, and should be active. Here are the conditions:

Rxn vol: 25ul
ATP (stock powder is dissolved in pH 7.2 buffer) final: 50uM
MBP (sigma cat# M1891): 5000ng/reaction
Antibody: anti-P-Thr from Cell Signalling cat# 9381

What can I do to reduce the initial ('no enzyme') MBP signal, and to generally improve the assay?

Thanks!]]> Thu, 19 Jan 2012 09:46:58 +0000 http://www.protocol-online.org/forums/topic/24082-kinase-assay-problem/ http://www.protocol-online.org/forums/topic/24061-tcaacetone-precipitation-no-visible-pellet-for-500-micrograms/
I am trying to develop some protein precipitation on BSA solution before using precious samples.

I am using 200 uL of 2.5 ug/uL of BSA in distilled water (solution not buffered). I have tried several things:

- add 400 uL of cold (-20C) TCA 10% in Acetone for 45 min ( so that's final concentration of 6.6%) at -20C
- centrifuge for 15 min at 21000g at 4C

OR

- add 1mL of cold (-20C) TCA 12% or 18% in Acetone overnight at -20C (so that's final concentrations of 10 and 15%)
- centrifuge for 15 min at 21000g at 4C

And I don't have any pellet at this time, I then wash twice with cold acetone and when I do a protein assay or a coomassie blue staining I don't have anything. Thus I believe I don't precipitate as I don't see a pellet or else the pellet is not visible and I discard it when I discard supernatant after centrifugation steps.

The step seems easy so I must do something wrong. Anyone could suggest something or identify where I could possibly go wrong?

Cheers,

Zakarino]]> Wed, 18 Jan 2012 09:53:40 +0000 http://www.protocol-online.org/forums/topic/24061-tcaacetone-precipitation-no-visible-pellet-for-500-micrograms/ http://www.protocol-online.org/forums/topic/24052-ultrasonic-bath-for-cell-lysis/
Do I need to take only cells or cells with pbs or with lysis buffer???

After Ultrasonic bath what shd i do for BCA protein analysis???

Thank you,
Vino]]> Tue, 17 Jan 2012 17:12:46 +0000 http://www.protocol-online.org/forums/topic/24052-ultrasonic-bath-for-cell-lysis/ http://www.protocol-online.org/forums/topic/24049-freeze-and-thaw-for-cell-lysis/
Do I need to take only cells or cells with pbs or with lysis buffer???

After freezing and thawing.. I should take the lysate???

Thank you,
Vinu]]> Tue, 17 Jan 2012 16:37:45 +0000 http://www.protocol-online.org/forums/topic/24049-freeze-and-thaw-for-cell-lysis/