Download loading control antibodies for western blotting

LOADING CONTROL ANTIBODIES FOR
WESTERN BLOTTING
by Deborah Grainger
The proteins and peptides that regularly serve as endogenous or internal controls
may not always take center stage in your research, but they are indispensable to your
conducting meaningful experiments and are essential for publication.
Western blotting requires such controls: it is widely used for the semi-quantification of protein levels
under of a set of different experimental parameters. Protein standards are required to make sense of
Western blotting results, and check that any increases and decreases in target proteins are actually due to
experimental manipulations and not, for example, because the sample went wandering during gel loading.
Internal control proteins — i.e. those with constant, unchanging levels — are usually detected in a second
round of blotting, following primary detection of your protein of interest. This step is used to standardize
results and normalize for any errors that creep into a Western blot experiment, such as sample loss through
loading at SDS-PAGE or Western blot transfer.
The loading control candidates for Western blotting are usually proteins with high and constitutive
expression. As mentioned above, the most basic criterion for a loading control is that its level remains
unchanged throughout an experiment, regardless of tissues or cell types used and how they are handled.
This means control candidates require careful selection; even bastions of the loading control repertoire —
such as β-actin and α-tubulin — can be affected by the conditions of your experiment (so be sure to double
check your chosen manipulations do not impact them.)
Here we’ve provided background information on each of the internal control proteins targeted by control
antibodies we offer, helping you choose the best control for your personal needs…
Actin
Type: Whole cell/cytoplasm
Molecular weight: ~42kDa
The six isofo ms of actin constitute a family of highly conse ved globula p oteins comp ised of th ee main
isofo m g oups, alpha, beta, and gamma. The alpha actins alpha C1 and alpha 1 and 2 a e a majo
constituent of the cont actile appa atus in muscle tissues. The beta (β) and gamma 1 and 2 (γ1 and γ2)
actins co exist in most cell types and a e an integ al pa t of the cytoskeleton; they a e mediato s of cell
t afficking, st uctu al integ ity and cell motility. Togethe , actins a e the most abundant p oteins in the
typical euka yotic cell, accounting fo about 15 pe cent of total p otein in some cell types. As such, actin is
widely used as an inte nal cont ol in Weste n blotting expe iments.
P oteintech’s polyclonal ACTB antibody (20536 1 AP) was gene ated using a β actin p otein antigen (amino
acids 14 167) and ecognizes all fo ms of actin, making it a pan actin antibody. Many studies use β actin
antibodies ecognizing all actin isofo ms to p obe fo this loading cont ol collective. Howeve , if you
studies involve wo k with skeletal muscle samples, o you a e wo king with conditions that see changes
in cell g owth o alte ed inte actions with the ext acellula mat ix, anothe loading cont ol may be bette
suited to you needs.
Actin antibodies
Related antibodies
ACTB
10081 224
Rabbit poly
ACTA1
10082 472
Rabbit poly
ACTB
10081 974
Mouse Mono
ACTA2
10155 850
Rabbit poly
HeLa cell lysate (10 ug/lane) was sepa ated
by SDS PAGE and actin was detected by
anti ACTB antibody 20536 1 AP at va ying
dilutions. (L R) 1:500, 1:1,000, 1:2,000
and 1:4,000.
LOADING CONTROL ANTIBODIES FOR WESTERN BLOTTING
COX-4
Type: Mitochondrial
Molecular weight: 17kDa
COX 4, o COX V (cytoch ome c oxidase subunit V), is a nuclea encoded subunit of the human
mitochond ial espi ato y chain enzyme cytoch ome c oxidase (COX). The COX 4 subunit can be exp essed
as eithe of two isofo ms, isofo m 1 and 2 named COX4 1 and COX4 2 espectively. COX4 1 exp ession is
ubiquitous th oughout all tissues, whilst COX4 2 is lung specific. Because of its dependably high level,
COX4 1 is commonly detected as an effective loading cont ol fo mitochond ial p oteins. Howeve , some
caution is advised when selecting this p otein fo Weste n blot detection as many othe p oteins un at
its 17kDa size du ing SDS PAGE (make su e you band of inte est won’t be obscu ed). t is also adviso y to
double check that any expe imental manipulations do not affect its levels. Fo an alte native mitochond ial
p otein loading cont ol see ou ent y on VDAC1 below.
P oteintech’s COX4 1 antibody (11242 1 AP) was gene ated against a COX4 1 whole p otein antigen
(amino acids 1 169) and also ecognizes COX4 2.
mmunop ecipitation of COXV f om mouse
skeletal muscle whole tissue lysate using
COXV antibody 11242 1 AP.
COX-4 antibodies
COX411
10085 086
Rabbit poly
COX412
10085 058
Rabbit poly
GAPDH
Type: Whole Cell/cytoplasmic
Molecular weight: 36kDa
Glyce aldehyde 3 phosphate dehyd ogenase, commonly known as GAPDH, catalyzes the sixth step of
glycolysis. t also pa ticipates in nuclea events such as t ansc iption, RNA binding and t anspo t, DNA
eplication and epai , as well as apoptosis. ts exp ession is high and constant in most tissues and cell types,
ea ning the gene and its p otein housekeeping status. Because of this, GAPDH is commonly used as a p otein
loading cont ol in Weste n blot and inte nal cont ol fo RT PCR. Howeve , the amount of GAPDH exp ession
can diffe between some tissues and its suitability fo you expe iment should be taken into conside ation
befo e selecting this ta get as a cont ol. t is also wo th noting that some physiological facto s, such as hypoxia
and diabetes, inc ease GAPDH exp ession in ce tain cell and tissue types.
P oteintech has both monoclonal GAPDH (60004 1 g) and polyclonal GAPDH (10494 1 AP) antibodies
available, both aised against a whole p otein antigen (amino acids 1 335) of human o igin.
GAPDH antibodies
GAPDH
10087 386
Rabbit poly
GAPDH
10087 384
Mouse poly
Weste n blot with Hela cell lysate using
anti GAPDH (10494 1 AP) at va ious dilutions
(L R: 1:2000, 1:4000, 1:8000 and 1:16000).
Lamin B1
Type: Nuclear
Molecular weight: 66kDa
Lamins a e integ al components of the nuclea lamina, a dense, fib ous laye unde lying the nuclea
envelope on its nucleoplasmic side. Lamins play an impo tant ole in the st uctu al integ ity of the nucleus
and its t affic cont ol, as well as inte acting with ch omatin and gene exp ession. Ve teb ate lamins consist
of two types, A and B. The LMNB1 gene encodes one of the two B type p oteins, lamin B1 and can be
used as a loading cont ol fo those wo king with nuclea f actions; howeve , this p otein is not suitable
fo samples whe e the nuclea envelope has been emoved. t is also wo thwhile to note that lamins
become phospho ylated du ing mitosis when the lamina mat ix is eve sibly disassembled. P oteintech’s
LMNB1 antibody (12987 1 AP) has been aised against a p otein antigen (amino acids 236 to 586 at the
C te minus) and is validated fo use in Weste n blot, HC, EL SA and immunofluo escence.
mmunofluo escence analysis of Lamin B1
in HepG2 cells, using LMNB1 antibody
12987 1 AP at 1:50 dilution and F TC labeled
donkey anti abbit gG ( ed).
Lamin antibodies
Lmnb1
Related antibodies
10089 490
Rabbit poly
LMNA/C
10089 486
Rabbit poly
LOADING CONTROL ANTIBODIES FOR WESTERN BLOTTING
PCNA
Type: Nuclear
Molecular weight: 36kDa
P olife ating Cell Nuclea Antigen (PCNA) is a p ocessivity facto fo DNA polyme ase δ; it helps cont ol
euka yotic DNA eplication by inc easing polyme ase nucleotide p ocessing ability du ing elongation of the
leading st and. PCNA p otein has been highly conse ved th oughout evolution the amino acid sequences
of ats and humans diffe by only 4 of 261 amino acids meaning whole p otein aised antibodies
ta geting PCNA should wo k ac oss multiple species.
Levels of PCNA do not va y with cell cycle status in mammalian cells, but, as it is mo e abundant in
p olife ating cells, PCNA is mostly used as loading cont ol in cell populations unde going p olife ation.
PCNA is best avoided if you expe iments induce DNA damage as this p otein is quickly deg aded when DNA
damage pathways a e activated.
mmunofluo escence analysis of PCNA
in HepG2 cells, using PCNA antibody
10205 2 AP at 1:50 dilution and F TC labeled
donkey anti abbit gG (g een).
P oteintech’s PCNA antibody is a abbit polyclonal antibody aised against an inte nal egion of human
PCNA, encompassing amino acids 8 256.
PCNA antibodies
PCNA
10091 936
Rabbit poly
PCNA
10091 934
Mouse poly
Tubulin
Type: Whole cell/cytoplasmic
Molecular weight: 50-55kDa
Tubulins a e the majo components of mic otubules, the majo t anspo t netwo k in cells. The mic otubules
a e involved in a wide va iety of cellula activities anging f om mitosis and t anspo t events to cell
movement and the maintenance of cell shape. Highly and stably exp essed and conse ved ac oss the
species, tubulins make excellent whole cell o cytoplasmic f action loading cont ols in Weste n blotting.
Howeve , tubulin exp ession may va y acco ding to esistance to antimic obial and antimitotic d ugs.
P oteintech has polyclonal antibodies against seve al tubulin subunits including an α tubulin antibody
(11224 1 AP) and two β tubulin antibodies 10068 1 AP and 10094 1 Ap.
Related antibodies
Tubulin antibodies
GAPDH
10081 874
Rabbit poly
10096 498
β tubulin(antigen:
amino acids 43 258)
Rabbit poly
10096 496
β tubulin (antigen:
amino acids 57 294)
Rabbit poly
α tubulin
10082 812
Mouse Mono
Weste n blot with Hela cell lysate using
anti GAPDH (10494 1 AP) at va ious dilutions
(L R: 1:2000, 1:4000, 1:8000 and 1:16000).
TBP
Type: Nuclear
Molecular weight: 38kDa
The TATA binding p otein (TBP) is a t ansc iption facto that binds specifically to the TATA box DNA
sequence, found a ound 25 30 base pai s upst eam of the t ansc iption sta t site of a ound 10 20 pe cent
of euka yotic gene p omote s. TBP, along with a va iety of TBP associated facto s, make up the TF D, a
gene al t ansc iption facto that in tu n makes up pa t of the RNA polyme ase p einitiation complex (P C).
As one of the few p oteins in the RNA P C that binds DNA in a sequence specific manne , it helps position
RNA polyme ase ove the t ansc iption sta t site of the gene. TBP is widely exp essed, though its levels a e
highest in the testis and ova y. This p otein is not a suitable standa d fo expe iments whe e DNA and othe
nuclea components have been emoved.
P oteintech has a polyclonal antibody available, aised against full length TBP binding p otein.
TBP antibodies
COLO 320 cells we e subjected to SDS
PAGE followed by weste n blot with
22006 1 AP(TBP antibody) at dilution
of 1:1000
TBP
10081 148
Rabbit poly
LOADING CONTROL ANTIBODIES FOR WESTERN BLOTTING
VDCA1/Porin
Type: Mitochondrial
Molecular weight : 31kDa
Voltage-dependent anion-selective channel protein 1 (VDAC1), also named porin 31HM,
porin 31HL or plasmalemmal porin, is the most widely expressed isoform of the eukaryotic
mitochondrial porin family. It forms a channel through the outer mitochondrial membrane
which adopts an open conformation at low or zero membrane potential and a closed
conformation at potentials above 30-40mV. VDAC1 is generally thought to be the principal
means by which metabolites diffuse in and out of the mitochondria and also has a secondary
role in apoptotic signaling. It is ubiquitously expressed throughout tissues, though its levels
are elevated in heart, liver and skeletal muscle tissues. It is conserved across the species
including, chimpanzee, dog, cow, mouse, rat, chicken, and zebrafish. Detection of VDAC1 is a
suitable mitochondrial loading control in whole-cell, cytoplasmic or mitochondrial extracts.
Proteintech has two rabbit polyclonal antibodies against VDAC1 available.
HEK293 cell lysate was sepa ated by SDS
PAGE followed by detection of VDAC1 by
Weste n blotting with P oteintech VDAC1
antibody 10866 1 AP at a dilution of 1:1000.
VDAC1 antibodies
VDAC1
10096 950
Rabbit poly
VDAC1
10096 948
Mouse poly
(Both aised against a whole p otein antigen: VDAC1 amino acid esidues 1 283.)
0615 Lit. No. 161294W