Can anyone suggest a good protein cell fractionation kit?

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Anyone know a good anti-phosphothreonine and anti-phosphoserine antibodies for IP and WB?

07 August 2012 1,769 10 View

Co-IP using crosslinking

I am trying to do a Co-IP, I think that my interactions is very weak and/or transient, because I can't see anything by regular Co-IP. Anyone use or know a protocol for Co-IP using crosslinking?

03 April 2012 7,134 8 View

Hiseq2000 vs Miseq What are the differences??

What do you choose for ChIP-seq? Thanks!

01 February 2012 5,608 2 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...

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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

Faye Stringer

Hi Loreto. Just to clarify: by wb do you mean western blot?

Jorge Martin-Perez

Loreto we were recommended by the Proteomic Service of the “Centro Nacional de Biotecnologia” (Consejo Superior de Investigaciones Cientificas, Madrid) to use ProteoExtract (Subcellular Proteome Extraction Kit) from Calbiochem (Cat# 539790). It is clear that when you collect the “membrane fraction”, it contains cellular membranes and not only plasma membrane. I hope this information is useful for you.

Best regards,

Jorge

Loreto Carvallo

Thanks guys!!!!

Faye yes, to do western blot

Gracias Jorge!

yes please! I appreciate!!! my email is [email protected]

and Do you know how it work (in the another lab)???