What is the transfer condition for handmade 1.5mm 15% gel to transfer histone H4?

I AM USING 20% METHANOL IN TRIS GLYCINE , 0.2 MICRON NITROCELLULOSE MEMBRANE FOR 100 V HR . the ponceau staining looks weaker compared to precast gel 10-12% with the transfer using sds and...

02 March 2018 327 3 View

Does molten magensium react with nickel curcible?

08 September 2014 7,456 4 View

Can we store Annexin V FITC stained cells and use after a week?

Can we stain the cells with annexin v fitc and fix with pfa and then store? If yes, can anyone suggest the optimized protocol for flow cytometry?

03 April 2014 8,449 6 View

Does the primary Ab and secondary Ab from different companies matter?

I am using cell signalling primary antibody and secondary antibody from sigma and substrate is NBT/BCIP. primary Ab dilution 1:1000 (3hrs rocking) sec Ab 1:10000 (45min rocking) primary ab...

02 March 2014 1,594 6 View

How do you add output styles in Endnote web?

I want to add the XXX journal output style in endnote web. In my endnote web only 21 output style are available, e.g. numbered, Ris, science, etc.

02 March 2014 6,054 12 View

What might be the reason for ghost bands in western blot?

What might be the reason for ghost bands in western blot? 1. I have used 0.5% skim milk overnight blocking 2. Primary antibody dilution 1:1000 3hrs rocking (3xwash PBST/ PBS) RT 3.Secondary...

01 February 2014 6,593 6 View

Why are bands are not visible in PVDF membrane after being stained with ponceau?

I have transferred gel in PVDF membrane and stained with ponceau but I couldn't see any bands on the membrane, instead, the whole PVDF membrane is turned into pink color and the color is not going...

11 December 2013 2,417 3 View

What is the difference between scrapping and trypsinising cells for extracting protein?

Is there any difference in protein content or alteration in phosphorylation of protein by using scrapping method and trypsin method for doing western blot?

10 November 2013 354 9 View

Why protein from SDS PAGE is not transferred onto nitrocellulose membrane?

While I'm transferring the protein from SDS gel to Nitrocellulose membrane only marker is getting transferred but not protein. I used 100v for 1hr to transfer. Can we reuse a transfer buffer? Is...

08 September 2013 8,118 21 View

Can we reuse a primary antibody which is diluted skimmed milk powder? If yes, can anyone suggest storage conditions?

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01 February 2013 6,862 9 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