hk2 cells culture

TieQiao Wu via methods%40net.bio.net (by TieQiao.Wu At baker.edu.au)
Wed Jan 31 01:48:26 EST 2007


        I have  been trying to overcome some problem regarding hk2(renal tubular epithelial cell line) cell cultures in 6-well plate recently. The cells in 6-well plate were cultured in DMEM F-12 medium with 10% FBS(fetal bovine serum) for 24 hours, washed with PBS once and then added F-12 medium with 1% FBS. But every time I notice that cells around the edge of well were not attached well or even disattached after PBS wash and I see quite some cells floating right after fresh medium addition.Such problem has never been seen in 10-cm petri dish so far. I am sure that the whole process is very gentle. Just wondering anybody faced such phenomena and would like to share your idea and how to deal with.

Thank very much for help. 

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Subject: Methods Digest, Vol 19, Issue 18


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Today's Topics:

   1. Re: Basic question about PMSF (Bean Long)
   2. Peattie DEPC sequencing and sequencing gels (Erich Chapman)
   3. Re: lysis buffer for membrane bounded protein
      (Dr Engelbert Buxbaum)
   4. Re: Regeneration of ELISA-Plates (Dr Engelbert Buxbaum)
   5. Re: Basic question about PMSF (Dr Engelbert Buxbaum)


----------------------------------------------------------------------

Message: 1
Date: Tue, 19 Dec 2006 14:07:10 +1100
From: Bean Long <ben.long At yourfinger.anu.edu.au>
Subject: Re: Basic question about PMSF
To: methods At net.bio.net
Message-ID: <4587575e$1 At clarion.carno.net.au>
Content-Type: text/plain; charset=ISO-8859-2; format=flowed

Dr Engelbert Buxbaum wrote:
> Breslauer wrote:
> 
>> Listen,
>> is it normal that when adding PMSF (250mM solution in MeOH) to cell
>> free extract from E.coli (broked by sonication and spinned 12000g for
>> 30min) to the final concentration of 0,5mM buffer becomes cloudly and
>> st become to precipitate (probably protein?).
> 
> No. At this concentration PMSF should not precipitate proteins, nor
> should it precipitate itself (provided that excessively high local
> concentrations are avoided during mixing). 

I was surprised to hear you had a stock of 250 mM.  I didn't think PMSF, 
even in methanol, was that soluble.  I usually make up a 170 mM stock in 
isopropanol.  Having said that, I'm sure that wouldn't be the problem. 
You may want to check one of the original reports on using PMSF as a 
protease inhibitor:

James, G. T. 1978. Inactivation of the protease inhibitor 
phenylmethylsulfonyl fluoride in buffers. Analytical Biochemistry 
86:574-579.

You might find some clues here.

My first thought was that the methanol was the problem but the final 
concentration is too low to really be a problem unless you have 
extremely high [protein] and relatively little mixing upon PMSF addition.

Hope this helps.

-- 
Bean

Remove "yourfinger" before replying


------------------------------

Message: 2
Date: Mon, 18 Dec 2006 11:22:40 -0800
From: "Erich Chapman" <echapma1 At uoregon.edu>
Subject: Peattie DEPC sequencing and sequencing gels
To: methods At magpie.bio.indiana.edu
Message-ID: <1166469760.531529.alphamail At mailapps1.uoregon.edu>
Content-Type: text/plain; charset="us-ascii"

Hi,

For about a month now I've been trying to sequence a 40mer RNA using direct dification as described by Peattie.  I am trying for the A specific reaction in which DEPC (diethyl pyrocarbonate) is used to first modify the N7 of A>G and subsequent aniline induced cleavage.  I realize that RNAse U2 cleaves a A residues but I need the chemical method to comment on the N7 of the A's and as far as I can tell there's only 1 RNAse U2 supplier (pierce).

When I run the sequencing gels I see specific cleavage occuring at A's, however the bands containing DEPC reactions migrate slower and cause -OH ladders and a T1 ladder to merge into them.  I had thought that electric fields, salt concentrations, dye concentration had caused this merging but am now convinced it is the influence of the carrier tRNA added to the DEPC reactions.  I can't find documentation of this anywhere but my lab notices that a sample with significantly more RNA can cause neighboring lanes to distort or simply cause the highly loaded lane to move slower.  I was wondering if anyone can confirm this.

Also if anyone had a modernized procedure for Peattie A specific sequencing it'd be much appreciated.

One more thing too... which I phosphorimage the resulting gels I see the expected smiling of the bands but the smiling extends into lanes where I didn't load sample, as if the radioactive RNA has move laterally in the gel.  I am curious if this could be due to the water added around the edges of the gel in order to pull it onto Whatman paper or if the phenomenon has another basis.

I'd greatly appreciate any advice.

Erich Chapman
Ph.D. Student
University of Oregon



------------------------------

Message: 3
Date: Mon, 18 Dec 2006 21:02:49 +0100
From: Dr Engelbert Buxbaum <engelbert_buxbaum At hotmail.com>
Subject: Re: lysis buffer for membrane bounded protein
To: methods At net.bio.net
Message-ID: <em6s5b$57d$02$1 At news.t-online.com>
Content-Type: text/plain; charset=iso-8859-2

Paras Anand wrote:

> Can someone tell me about the best way to remove proteins that are 
> loosely associated to the plasma membrane.

Classical extraction media are 250 mM KI (Michelis & Spanswick, Plant
Physiol. 81 (1986) 542-7) or 100 mM Na2CO3 (Fujiki et al., J. Cell Biol.
93 (1982) 97-102).


------------------------------

Message: 4
Date: Mon, 18 Dec 2006 21:02:45 +0100
From: Dr Engelbert Buxbaum <engelbert_buxbaum At hotmail.com>
Subject: Re: Regeneration of ELISA-Plates
To: methods At net.bio.net
Message-ID: <em6s58$577$02$1 At news.t-online.com>
Content-Type: text/plain; charset=iso-8859-2

peter willingmann wrote:

> Subject said it all..  We want to use ELISA PLATES and /or strips for a 
> class demonstration/experiment. But the Plates/Strips are a little bit 
> expensive and we want to reuse again. How do you remove the coated 
> protein from the plate? HCL???

A typical question from a university. It should of course be possible to
strip adsorbed proteins in the same way as one uses in experimental
context, i.e. SDS in an acidic glycine-buffer, 1 M or so. But the costs
of the chemicals involved, and even more of the labour required, is much
higher than that of new plates. 

You are supposed to prepare students for a productive life, so don't
give a bad example. 


------------------------------

Message: 5
Date: Mon, 18 Dec 2006 21:02:51 +0100
From: Dr Engelbert Buxbaum <engelbert_buxbaum At hotmail.com>
Subject: Re: Basic question about PMSF
To: methods At net.bio.net
Message-ID: <em6s5e$f8p$01$1 At news.t-online.com>
Content-Type: text/plain; charset=iso-8859-2

Breslauer wrote:

> Listen,
> is it normal that when adding PMSF (250mM solution in MeOH) to cell
> free extract from E.coli (broked by sonication and spinned 12000g for
> 30min) to the final concentration of 0,5mM buffer becomes cloudly and
> st become to precipitate (probably protein?).

No. At this concentration PMSF should not precipitate proteins, nor
should it precipitate itself (provided that excessively high local
concentrations are avoided during mixing). 


------------------------------

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