crom80

Hi,

Recently there was a publication that a human cancer cell line we use in the lab is contaminated with mouse cells. Some other cell lines we also use from the same institution we recieved the contaminated cell lines we found were pulled from ATCC web site. We are fearing the worst and want to quickly test all cell lines to check for mouse cell contaminations.

Is there a quick kit or method to test? We were thinking of PCRing a mouse gene or something along those lines. Commercial kits seem very expensive.

Any help would be great.

I already lost half of my data for a paper from losing that cell line. I don't want to lose the other half...

Hi,

I am looking at the localization of Protein X during UV irradiation.
I noticed that increased fluorescence of protein X using a monoclonal antibody within 30 minutes of UV irradiation. Since the increase is in a very short time period I do not think it is due to increased expression or decreased degredation (if so, that itself would be a lone paper).
I am hypothesizing that since I used a monoclonal antibody, UV irradiation caused a shift in protein folding or uncovering of the antigen binding site, hence the increase in immunofluorescence signal.
So I am planing to stain with a polyclonal antibody.

I was wondering, if I simultaneously incubate the samples with mouse polyclonal and rabbit monoclonal to the same protein, would I be too optimistic in thinking I would see good staining? Should I just do the two antibodies separately?
And if I go ahead with simultaneous staining, should incubate the two primary antibodies at the same time or one after the other? (monoclonol first or polyclonol first?)
I tried to search the literature for any suggestions but I could not find any attempts such as this.

Thank you.

Hi,

I am trying to assay cMet-phosphorylation during HGF treatment in HeLa cells.

 Untitled-1.jpg   43.13K   75 downloads

I have attached a figure of my attempted assay. The positive control is an HCC827 cell extract (ready to be loaded) graciously sent from a company as I was troubleshooting (hence, I did not lyses and prepare sample for western). As seen in figure 10-30 minutes after HGF treatment show some increase in phospho-cMet (primary AB: Cell signaling Cat# 3077) but may be due to the slight increase in total cMet (primary AB: Cell signaling Cat# 8198). So I also examined ERK phosphorylation and I see a better readout for HGF signaling suggesting the HGF I am using has not gone bad.

Below is my lysis/sample buffer and protocol.

My lysis buffer is:
-50mM Tris HCl pH 7.4
-150mM NaCl
-0.5% sodium deoxycholate
-1% TritonX-100
-0.1% SDS
-1mM EDTA
Below are added prior to lysis
-1:100 of Sigma Protease inhibitor cocktail
-10mM Na3VO4
-40mM beta-glycerolphosphate
-20mM NaF

my 5xsample buffer is:
-0.25M Tris HCl (pH6.8)
-10%SDS
-0.5%Bromophenol Blue
-50% Glycerol
-2ml beta-mercaptoethanol.


- I serum starve ~90% confluent HeLa cells for atleast 8 hours - 12 hours.
- Replace media on cells with serum free media + 50ng/ml HGF.
- After indicated time (2-30 min) I wash once with cold PBS (-Ca/Mg)
- add cold lysis buffer and shake briefly to make sure monolayer is covered and does not dry out as I scrape.
- Scrape and place cells in tube on Ice (I flush in and out with pipette as I place in tube).
- Vortex briefly every 10 minutes and place on Ice for a total 30 minutes.
- Vortex and centrifuge 15k rcf for 15 min at 4C.
- Collect supernatant
- Measure concentration (I get around 2-4 ug/ul of protein from 6-well plates without sonication so not too bad). Also I may save samples at this step at -20C if I plan to use samples within 3 days and at -80C for longer storage.
- Bring concentration of samples to equal amounts with 5X sample buffer and the lysis buffer+inhibitors as dilution.
- boil sample 5 min at 95-100C and place on ice.
- Run on gel and transfer.

The main focus of the protocol is try to perform everything as quickly as possible and keep on ice (no ice bucket in hood though). As seen with ERK phosphorylation it seems my stimulation and collecting methods aren't bad.

I'm wondering if my lysis/sample buffer or method of preparing loading samples are bad for examining this type of phosphorylation (membrane associated receptor with epitope specific phopho primary AB)

Any suggestions are appreciated.

 WB.jpg   177.95K   128 downloads

Hi, long time browser first time poster.

I have attached an image of my western blot membrane. Top two membranes are phopho-cMet and bottom two are total c-Met. The exposure is 30 seconds.

I have been following the protocol given on the antibody company's webpage (cell signaling) and other papers have used the same antibodies with clear results. I have also been calling/e-mailing tech support from the company trying to figure out why my background is so high.

Below is my protocol (also cell signaling company's) from transfer to exposure:

- Wet Transfer of 10% gel to PVDF (already activated by methanol and washed in dH20 for 5 min) at 78V for 2 hours.
- Place transfered membrane (no air/methanol/heat drying) in 5% non-fat dry MILK (have tried BSA as well and was told by company to do Milk) TBS-T(0.05% Tween-20) for 1 hr at room temp with agitation.
- wash three times 5 min each in TBS-T
-Incubate in primary 1:1000 5%Milk for total and 5%BSA for phospho TBS-T overnight at 4C (for those who have used the same antibodies and may have suggestions, Cat# 8198 and 3077)
- wash three times 5 min each in TBS-T
- incubate in secondary 1:2000 in 5% Milk for both total and phospho TBS-T 1 hr at room temp.
- wash three times 5 min each in TBS-T
- ECL 1 min
- expose to film.

In addition to this I have tried additional and longer washes after initial ECL/exposure and high salt wash with the same TBS-T(0.05% Tween-20)+0.5M NaCl with not much improvement.

I have tried with freshly made transfer and wash buffers in case of contamination issues. Same membrane probing with ubiquitious ERK total and phospho antibodies show up nicely.

Greatly appreciate any suggestions!!!