---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2008-11-13 18:40:21+00:00
layout: post
slug: western-blot-purified-his-column-decorin-fst-lap-telethonin
title: Western Blot - Purified (His column) decorin, FST, LAP & telethonin
categories:
  - 2008
  - Myostatin Interacting Proteins
tags:
  - anti-His tag
  - chemiluminescent
  - Coomassie
  - decorin
  - follistatin
  - FST
  - LAP
  - Oncorhynchus mykiss
  - rainbow trout
  - SDS-PAGE
  - telothonin
  - Western blot
---

10uL of each sample from yesterday (binding supe, washes and elution fractions) were mixed with 2x sample reducing buffer and boiled 5 mins. Samples were loaded onto Pierce 4-20% Tris-Glycine SDS/PAGE gels. 10uL of SeeBlue ladder was loaded. 5uL of positive control lysate was loaded. Gels were run @ 150V for 45mins.

Gels were transferred to nitrocellulose membrane (equilibrated in tris-glycine transfer buffer for 5mins) @15V for 15mins. Membrane washed briefly in 1x TBS-T, then blocked for 1hr. Gel was stained for 1hr in Coomassie stain and then destained w/ 10% acetic acid until bands were clearly visible.



### Gel #1 - Decorin samples



![](https://eagle.fish.washington.edu/Arabidopsis/SDS-PAGE/20081114-01.JPG)

Lane #1 - Ladder

Lane #2 - Positive control lysate

Lane #3 - Binding solution

Lane #4 - Wash

Lane #5 - Elution fraction #1

Lane #6 - Elution fraction #2

Lane #7 - Elution fraction #3

Lane #8 - Elution fraction #4

Lane #9 - Elution fraction #5

Lane #10 - Elution fraction #6

Lane #11 - Elution fraction #7

Lane #12 - Elution fraction #8





### Gel #2 - FST samples



![](https://eagle.fish.washington.edu/Arabidopsis/SDS-PAGE/20081114-02.JPG)

Lane #1 - Binding solution

Lane #2 - Wash

Lane #3 - Ladder

Lane #4 - Positive control lysate

Lane #5 - Elution fraction #1

Lane #6 - Elution fraction #2

Lane #7 - Elution fraction #3

Lane #8 - Elution fraction #4

Lane #9 - Elution fraction #5

Lane #10 - Elution fraction #6

Lane #11 - Elution fraction #7

Lane #12 - Elution fraction #8





### Gel #3 - LAP samples



![](https://eagle.fish.washington.edu/Arabidopsis/SDS-PAGE/20081114-03.JPG)

Lane #1 - Ladder

Lane #2 - Positive control lysate

Lane #3 - Elution fraction #1

Lane #4 - Elution fraction #2

Lane #5 - Elution fraction #3

Lane #6 - Elution fraction #4

Lane #7 - Elution fraction #5

Lane #8 - Elution fraction #6

Lane #9 - Elution fraction #7

Lane #10 - Elution fraction #8

Lane #11 - Binding solution

Lane #12 - Wash





### Gel #4 - Telethonin samples



![](https://eagle.fish.washington.edu/Arabidopsis/SDS-PAGE/20081114-04.JPG)

Lane #1 - Binding solution

Lane #2 - Wash

Lane #3 - Ladder

Lane #4 - Positive control lysate

Lane #5 - Elution fraction #1

Lane #6 - Elution fraction #2

Lane #7 - Elution fraction #3

Lane #8 - Elution fraction #4

Lane #9 - Elution fraction #5

Lane #10 - Elution fraction #6

Lane #11 - Elution fraction #7

Lane #12 - Elution fraction #8



Results:

Gels all have protein, but no elution fractions exhibit just a single band. Additionally, most of the samples across the different genes show similar banding patterns.







Primary Ab (anti-6x His) was added to membrane at a 1:15,000 dilution. This incubated for 1hr. Used Ab solution was stored @ 4C.

Membrane was washed with 1x TBS-T and fresh blocking solution was added per the [protocol](/protocol#tocWestern).

Secondary Ab (DAR HRP) was added to membrane @ 1:15,000 dilution and incubated for 30mins.

Membrane was washed with 1x TBS-T per the [protocol](/protocol#tocWestern).

Blots were developed using Millipore Immobilon chemiluminescent regent and imaged.



Results:

NONE! All four membranes are completely blank. Not even a signal from the positive control lysate. Infuriating! Why? Transfer was performed in the correct orientation (this was confirmed by the presence of the ladder dye on the membranes post-transfer). The same Ab stocks and developer stock were used as a couple of weeks ago, so these shouldn't be a concern. Ab was definitely added to all the samples, so that's ruled out. Ugh.
