Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Protein Science > Western Blot Forum
Register Search Today's Posts Mark Forums Read

Western Blot Forum Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


Western blot mystery - possibly a sample preparation problem?

Western blot mystery - possibly a sample preparation problem? - Western Blot Forum

Western blot mystery - possibly a sample preparation problem? - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 03-29-2012, 03:37 AM
Pipette Filler
Points: 5, Level: 1 Points: 5, Level: 1 Points: 5, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Mar 2012
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Default Western blot mystery - possibly a sample preparation problem?



Hello!

I've been trying for a few weeks to run some western blots in my lab, with limited success. I really need to get it working (my PI is getting very annoyed with me), but I'm having a hell of a time figuring out why it isn't working. I'm the only one in my current lab running westerns, so I'd really appreciate some help!

The samples I'm using are from total cell lysates of rat spleens, mouse thymus, and MCF10A human cells. I've been able to detect actin and tubulin, though the detection isn't as high as I would like. That may be due to an old antibody, though. The bigger problem is that I haven't been able to detect Bcl-2 in my samples. It's a smaller protein (~25kDa), which may be part of the problem. Using a positive control mouse spleen sample from Santa Cruz Biotech (where my antibody is from), I've gotten a Bcl-2 signal, so that suggests it has something to do with how my samples are prepared.

I'm preparing my samples according to a protocol I found (******** and Nister 2003), briefly: cells are lysed in SDS (basically laemmli buffer without bromophenol) by pipetting and vigorous vortexing, then incubating at 70 degrees for 20 minutes. Quantification of my samples by BioRad RC/DC puts them at about 2mg/mL. I've been loading 20ug per lane (15uL well sizes, but I don't like overfilling them).

An observation that may be related is that the blue line in the running gel runs very quickly through to the bottom, and gets very dispersed. The bottom few markers on my protein ladder are also smearing. I've tried using gels from 8% to 12%, and varying the voltage from 125 to 200, and nothing seems to have an effect. I've ordered some pre-cast 4-20% gels to see if that helps.

Does anyone have any ideas what may be going wrong? I've been frustrated with this for far too long, this *should* be a real simple, quick assay and I should have been done with it weeks ago. I think my PI is going to be very annoyed if I don't have any data for him before he leaves for AACR this weekend!

TIA

Mark
Reply With Quote
  #2  
Old 07-12-2012, 07:48 PM
Pipette Filler
Points: 44, Level: 1 Points: 44, Level: 1 Points: 44, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2012
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Question Re: Western blot mystery - possibly a sample preparation problem?

Hi

I am totally new in the field of research.

I have been trying to get some good results but so far, I have been unsuccessful. The gels I prepare give me a strange bluish hue in the bottom gel area. I have tried to change ingredients and prepared fresh Tris buffer to prepare the gels but it still looks the same way. The top gel is OK though.

After I am done and go for detection, I usually get a lot of non specific binding on the film. Also, some times I get nothing at all. Most of the time there are blurrs here and there with no bands. I don't know If its the problem with gels or antibodies. I am also thinking of preparing the ECL Buffer fresh. Please give me any other suggestions that can help. I shall be thankful.
Reply With Quote
Reply

Tags
blot , mystery , possibly , preparation , problem , sample , western


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
Western Blot - How to Load Equal Amounts and Volumes of Protein? shawna82 Molecular Biology Techniques 0 02-27-2012 08:02 PM
How to Run a Concentrated Protein Sample for Western Blot? eng Western Blot Forum 1 02-18-2011 12:13 AM
Western blot admin Article Discussion 2 04-23-2010 07:31 PM
Western Blot Woes - Very Clean Western kiki06 Western Blot Forum 3 07-22-2008 02:30 PM
No Bands on Western Blot clokke76 Western Blot Forum 2 08-27-2007 04:26 PM


All times are GMT. The time now is 02:26 AM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.12436 seconds with 16 queries