Oct 16, 2019

Public workspaceWestern Analysis used in Oxidative Stress Protocols V.2

DOI
dx.doi.org/10.17504/protocols.io.8bbhsin
  • Eva Feldman1
  • 1University of Michigan - Ann Arbor
  • Diabetic Complications Consortium
    Tech. support email: rmcindoe@augusta.edu
Lili Liang
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DOI: dx.doi.org/10.17504/protocols.io.8bbhsin
External link: https://www.diacomp.org/shared/document.aspx?id=31&docType=Protocol
Protocol Citation: Eva Feldman 2019. Western Analysis used in Oxidative Stress Protocols. protocols.io https://dx.doi.org/10.17504/protocols.io.8bbhsin
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 16, 2019
Last Modified: October 16, 2019
Protocol Integer ID: 28739
Keywords: Biochemical Measures of Neuropathy, diabetic neuropathy, Western Analysis
Abstract
Summary:

This is the general protocol used for western analysis of samples from the Oxidative Stress Protocols. There are no specific antibodies described for use, rather a general procedure for creating the western. See the specific assay for the details about the antibodies used.



Diabetic Complications:



Materials
MATERIALS
Reagent 12.5% Acrylamide gel stock solution Merck MilliporeSigma (Sigma-Aldrich)
Reagent10% Ammonium persulfate (APS)Merck MilliporeSigma (Sigma-Aldrich)
ReagentTEMEDMerck MilliporeSigma (Sigma-Aldrich)
Reagent2-propanol Merck MilliporeSigma (Sigma-Aldrich)
Reagent10X TBS Merck MilliporeSigma (Sigma-Aldrich)
Reagent10x Sample Buffer
Reagent10X Running Buffer
Reagent10X Transfer Buffer
Reagent5% Fat Free Milk Catalog #Grocery Store
ReagentGel loading tipsFisher Scientific
ReagentNitrocellulose Schleicher & Schuell
ReagentECL kitAmersham plc
Reagent Preparation:



Separating Gel Preparation:

1 gel: 10mL 12.5% gel stock
50µL 10% APS
7µL TEMED

Stacking Gel Preparation:

2 gels: 5mL stacking gel stock
30µL 10% APS
5µL TEMED

Wash & dry plates.
Assemble rig and fill plates with H²O to check for leaks.
Pour off water and wipe dry with kimwipe.
Load gel to about the top of the door.
Add 2-propanol to cover the edge.
Wait ~ 40 minutes to polymerize.
Thaw samples on ice.
When gel is ready, pour off 2-propanol and rinse with H²O.
Remove excess H²O with a kimwipe.
Prepare and load stacking gel and insert comb making sure there are no bubbles under the teeth.
Put a beaker of water on the hot plate to boil.
Prepare samples:

♦ Plasma - dilute 2µL plasma in 198µL (1:50) RIPA buffer + inhibitors in a labeled screw top tube. Sonicate on 5. Pull off 10µL for Protein analysis. Add 38µL 10X samples buffer to the 190µL lysate.

♦ DRG and Sciatic nerve - DRG - After removing 4 DRG for TRAP assay, pool the remaining DRG in a labeled screw top tube. Sciatic nerve - Place 1 sciatic nerve into a labeled screw top tube.

Add 110µL RIPA buffer + inhibitors. Sonicate on 8 on ice. Freeze samples, thaw and run through a 1mL syringe with a 26g needle. Repeat Freeze, thaw and running through syringe. Pull off 10µL for Protein analysis. Add 20µL 10X sample buffer to the 100µL lysate.
Label screw top tubes for markers.
Do protein analysis on samples. Generally load 20 to 50µg
Add 2µL 10X sample buffer to 10µl rainbow protein marker. (times x for x # of gels)
Boil samples and markers for 5 minutes and cool.
When gel is done gently remove combs.
Assemble rig with short plate on the inside, press down and close doors.
Fill inside chamber with running buffer to about ½ way between top of sm & lg plate and make sure there are no leaks.
Pour more running buffer into outside of rig to the bottom of the gate.
Load rainbow protein marker and samples.
Set volts @ 200 and run for 50-60 minutes.
Remove gel from rig, remove wells and soak gel in transfer buffer for 15 minutes.
Cut and label nitrocellulose membrane to size and soak in transfer buffer.
In another dish, soak 2 fiber pads and 2 pieces of whatman paper for each gel.
Assemble the sandwich with black side down in transfer buffer, making sure there are no bubbles between each layer put 1 fiber pad, 1 whatman paper, gel, nitrocellulose, 1 whatman paper, and 1 fiber pad.
Put a stir bar in the bottom of the rig and place the sandwich in the transfer unit with the black part in the back. (Protein runs from black to red, to the membrane)
Fill the ice pack and place behind the sandwich.
Fill the unit with 1X transfer buffer until the ice pack floats or the top of the lower ledge.
Transfer at 100V for 1 hour (100kd-30kd) or 69V for very low proteins
Rinse the membrane in 1X TBS for 10 minutes.
Block overnight @ 4º or at RT for 2 hours in TBST/milk for polyclonal antibodies or TBST/BSA for mAbs.
Quick rinse once with TBST.
Incubate 2 hours at RT or overnight @ 4º in primary antibody in TBST/milk or TBST/BSA on rocker. (Primary antibody can be re-used)
Wash 3x’s for 5 minutes with TBST.
Incubate for 2 hour in secondary antibody in TBST/milk or TBST/BSA.
Quick rinse once with TBST.
Wash 3x’s for 5 minutes each in TBST.
Wash 20 minutes in 1X TBS.
In a 15mL conical tube, develop with small cell signaling bottles using 9mL H²O and 500µL of each reagent. Expose for 1 minute.
Develop film.