Sample preparation in PCR machine for SDS-PAGE?

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Query related to JC-1 mechanism

JC-1 dye is well known for using mitochondrial membrane potential disruption detection during apoptosis. JC-1 entry is from the cell membrane to the cytosol to the mitochondria and forms...

10 November 2012 4,939 15 View

Is it possible to use Alamar blue to test cell cytotoxicity at different time points with same sample ?

All these days we were using MTT for cell cytotoxicity. we purchased alamar blue from invitrogen newly, how can we use same 96 well plate reading after 1 day,2 day,3 days. Please share your...

08 September 2012 7,472 5 View

Is it possible to use Alamar blue to test cell cytotoxicity at different time points with the same sample ?

So far we've been using MTT for cell cytotoxicity. We purchased alamar blue from invitrogen newly: how can we use same 96 well plate reading after 1 day,2 day,3 days? As we know alamar blue is...

08 September 2012 9,000 3 View

Does an increase in caspase activity mean apoptosis should increase?

PI3k inhibitor(LY294002) and our test compound showed a 7 fold increase in caspase 3/7 activity . We further tested apoptosis by annexin V FITC+PI by flow cytometry. We found that in the PI3k...

07 August 2012 4,095 14 View

My HepG2 cell line is growing very slowly, why?

I am trying to grow hepatic cell lines like HepG2, HUH-7 and ,Hep-3b for anticancer work. I am growing all in RPMI-1640 + 10%FBS + 1X Ab/AM. HUH-7, and hep-3b are growing very nicely but HepG2...

03 April 2012 4,309 10 View

how to calculate IC50 using sigma plot software.

I do MTT assay to see the cell viability upon drug treatments.we have sigma plot.10 software programme.could anyone tell how to calculate IC-50 using that.

01 February 2012 8,601 5 View

Solvent for partially purified product?????????

We have some plant product partially purified powdered form,i need to check anticancer activitiy of that.iam dissolving product in Methanol and further dissolving in growth medium for MTT...

11 December 2011 6,473 3 View

Cleaved caspases detection

I would like to detect cleaved caspases (17,19 kd) so Is 15% SDS PAGE ok for this? running SDS PAGE 25mA for 1hr and western transfer conditions i use 100V for 1hr. and any suggestions during...

11 December 2011 3,606 1 View

Will a 10xHis-tag prevent anti-MBP from binding to MBP?

I have a protein with a 10xHis-MBP tag on the N-terminus. I'm fairly certain I am expressing my protein, but I'm not getting a signal on my western blot. Is there a possibility that the 10xHis tag...

25 February 2021 8,404 3 View

What's the concentration recommend for recombinant protein use in western blotting?

I'm currently working having problem with the concentration for recombinant protein use in western blot, the one been used is Recombinant Human CD19 Fc Chimera Protein-rndsystem which does not...

24 February 2021 8,514 3 View

What is the best way to manage Western blots?

Increasingly, more articles are being retracted because of duplicate or otherwise dubious Western blots. Consequently, journals are asking for original Western blots and authentication of...

23 February 2021 8,609 3 View

How to manage western blot for proteomics data validation?

Hi Everyone, I am a bigginer in western blotting and I am trying to use it for validation of my proteomics data. I have: two regulated proteins with molecular mass of 40 and 73 kDa one control...

23 February 2021 6,671 4 View

Diltuions for a Western blot?

I need help using the equation C1V1=C2V2 figuring out how much antibody to add to use for a western blot. The stock was origianlly 3.45 ng/ul but diluted 1:10 so now its 0.345 ng/ul. I need to run...

22 February 2021 9,901 3 View

Can anyone help me how to get the similar bands in Actin?

I run western blot on HCT-116 cell lines, I got actin band like in the attached picture. The band size is not same. Can anyone help me explain this situation and how to overcome? Thank you your...

22 February 2021 6,937 3 View

I see 2 random bands in my ERK/pERK blot? Has anyone else seen this?

I ran a western blot using 12% gel and probing for ERK/pERK (cell signaling antibodies). The green is total ERK, the Red is supposed to be phospho-ERK. I see 2 bands below the total ERK that...

22 February 2021 1,777 3 View

Will SARS-CoV-2 Spike be broken into S1 and S2 by denaturing conditions?

Hello, I am performing a western blot of my SARS-CoV-2 Spike-pseudotyped lentiviral particles. I denature my virus by adding concentrated virus to 2X Laemmli buffer at 1:1, then boiling for...

17 February 2021 4,844 4 View

Unable to detect a truncated protein variant, but I can observe a truncated cDNA from Sanger?

Hi, so basically... I am trying to mimic a clinically observed condition wherein a frame-shift mutation happens only at one allele of the patients, giving rise to a truncated variant which evades...

17 February 2021 1,130 6 View

(western blot) can methanol damage my antibody signal?

I accidentally rinsed with methanol after adding my primary antibody to my membrane. I noticed after ~10 seconds and immediately replaced it with PBST (3x15 min washes). Will my antibody signal be...

12 February 2021 5,824 3 View